Topical compositions and methods of using the same

ABSTRACT

The present invention generally relates to topical compositions and methods of using the same. A preferred topical pharmaceutical composition comprises a nitric oxide-releasing active pharmaceutical ingredient in admixture with a hydrophilic composition and a hydrophobic composition wherein the nitric oxide-releasing active pharmaceutical ingredient comprises a nitric oxide-releasing compound having a diazeniumdiolate functional group.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Contract No.W81XWH-11-C-0029 awarded by the U.S. Department of Defense and Grant No.5R43AI096569 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention generally relates to topical compositions andmethods of using the same.

BACKGROUND

Moisture sensitive active pharmaceutical ingredients (APIs) can presentchallenges when delivered topically. While the API may be incorporatedin a hydrophobic topical composition so that it has suitable shelf lifestability, this same stability may reduce the ability to deliver thedrug if moisture is the activating agent.

The present invention may address previous shortcomings in the art byproviding topical compositions and methods of using the same.

SUMMARY

A first aspect of the present invention comprises a topicalpharmaceutical composition comprising a nitric oxide-releasing activepharmaceutical ingredient in admixture with a hydrophilic compositionand a hydrophobic composition.

A second aspect of the present invention comprises a compositioncomprising: an active pharmaceutical ingredient; a hydrophobic base; atleast one of an amphiphilic compound or an emulsifying agent; a buffer;a polymer, a polyhydric alcohol; and water, wherein the composition isbuffered to a pH of about 3 to about 9.

A further aspect of the present invention comprises a composition fortopical administration comprising a self-emulsifying admixture.

Another aspect of the present invention comprises a kit comprising: afirst composition comprising a hydrophilic composition; and a secondcomposition comprising an active pharmaceutical ingredient.

An additional aspect of the present invention comprises a method ofincreasing the release of nitric oxide from a hydrophobic compositioncontaining a diazeniumdiolate modified macromolecule comprising:admixing a hydrophobic composition with a hydrophilic composition havinga pH of about 4 to about 8 to form an admixture; and applying theadmixture to the skin of a subject.

A further aspect of the present invention comprises a method ofproviding a topical antimicrobial composition comprising: admixing ahydrophobic composition with a hydrophilic composition having a pH ofabout 4 to about 8 to form an admixture; and applying the admixture tothe skin of a subject.

Another aspect of the present invention comprises a method of increasingthe rate of healing for a wound comprising: applying topically anadmixture having a pH of about 5 to about 8.

The foregoing and other aspects of the present invention will now bedescribed in more detail including other embodiments described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph of the NO release from different formulationscontaining Nitricil™ NVN4 ointment (1.8% w/w NO).

FIG. 2 shows a graph of the combined MRSA Staphylococcus aureus ATCC BAA1686 bacterial counts after treatment application on days 4 and 7.

FIG. 3 shows a graph of the combined MRSA Staphylococcus aureus ATCC BAA1686 bacterial counts after treatment application on days 4 and 7.

FIG. 4 shows a graph of the A. baumannii bacterial counts aftertreatment application on days 4 and 7.

FIG. 5 shows a graph of the S. aureus bacterial counts after treatmentapplication on days 4 and 7.

FIG. 6 shows a graph of the C. albicans bacterial counts after treatmentapplication on days 4 and 7.

FIG. 7 shows a graph of the cumulative nitric oxide (NO) release overtime for each of the three admixtures.

FIG. 8 shows a graph of the real time NO release over time for each ofthe three admixtures.

FIG. 9 shows a graph of the cumulative nitric oxide (NO) release overtime for each of the two ointments.

FIG. 10 shows a graph of the real time NO release over time for each ofthe two ointments.

FIG. 11 illustrates an example schematic of oil droplets encompassing anAPI with water surrounding the oil droplets.

DETAILED DESCRIPTION

The present invention will now be described more fully hereinafter. Thisinvention may, however, be embodied in different forms and should not beconstrued as limited to the embodiments set forth herein. Rather, theseembodiments are provided so that this disclosure will be thorough andcomplete, and will fully convey the scope of the invention to thoseskilled in the art.

The terminology used in the description of the invention herein is forthe purpose of describing particular embodiments only and is notintended to be limiting of the invention. As used in the description ofthe invention and the appended claims, the singular forms “a”, “an” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the present applicationand relevant art and should not be interpreted in an idealized or overlyformal sense unless expressly so defined herein. The terminology used inthe description of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. In case of a conflict in terminology, the presentspecification is controlling.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

Unless the context indicates otherwise, it is specifically intended thatthe various features of the invention described herein can be used inany combination. Moreover, the present invention also contemplates thatin some embodiments of the invention, any feature or combination offeatures set forth herein can be excluded or omitted. To illustrate, ifthe specification states that a complex comprises components A, B and C,it is specifically intended that any of A, B or C, or a combinationthereof, can be omitted and disclaimed.

As used herein, the transitional phrase “consisting essentially of” (andgrammatical variants) is to be interpreted as encompassing the recitedmaterials or steps “and those that do not materially affect the basicand novel characteristic(s)” of the claimed invention. See, In re Herz,537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in theoriginal); see also MPEP §2111.03. Thus, the term “consistingessentially of” as used herein should not be interpreted as equivalentto “comprising.”

The term “about,” as used herein when referring to a measurable valuesuch as an amount or concentration and the like, is meant to encompassvariations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specifiedvalue as well as the specified value. For example, “about X” where X isthe measurable value, is meant to include X as well as variations of f10%, ±5%, ±1%, f 0.5%, or even f 0.1% of X. A range provided herein fora measureable value may include any other range and/or individual valuetherein.

According to some embodiments of the present invention, provided hereinare topical compositions. In some embodiments, a topical composition ofthe present invention comprises an admixture. “Admixture” as used hereinrefers to a combination of at least two different compositions. In someembodiments, the at least two different compositions may be miscible. Inparticular embodiments, the term admixture refers to the at least twodifferent compositions being maintained substantially isolated from oneanother until the proximate time of use or application. In certainembodiments, the term admixture refers to the at least two differentcompositions being maintained substantially isolated from one anotheruntil dispensing, such as with pharmacist dispensed products. In someembodiments, one or more compositions present in an admixture may bemaintained substantially isolated from one or more other compositionspresent in an admixture. The term admixture is not intended to refer toa composition that is created at the time of manufacture of thecomposition or product, such as by the combining of ingredients tocreate the composition. The combining of two or more differentcompositions, such as 2, 3, 4, 5, 6, 7, or more compositions, to form anadmixture may be achieved by mixing, blending, contacting, applying to asame area or region, emulsifying, and the like the two or more differentcompositions. The combining of two or more different compositions may becarried out to induce a chemical reaction. A composition may bedifferent than another composition in the amount or concentration of oneor more components, the type (e.g., chemical composition) of one or morecomponents, and/or the presence and/or absence of one or morecomponents.

An admixture of the present invention may comprise at least onecomposition that modulates a property of another composition and/or acomponent present in the admixture. The property modulated may becompared to the property of the composition and/or component in theabsence of the admixture. For example, the admixture may comprise atleast one composition (i.e., a first composition) that modulates the pHof another composition (i.e., a second composition) and/or the releaseof an active pharmaceutical ingredient (API) in another composition(i.e., a second composition). As used herein, release of the API refersto release of the API itself and/or release of one or more active agentsfrom the API (e.g., where a pro-drug is the API and the active form ofthe drug may be released). For example, in embodiments where the API isa nitric oxide-releasing API, references to API release may refer torelease of nitric oxide from the API. The pH of the admixture may becompared to the pH of the second composition when it is not in admixturewith the first composition. The release of the API from the admixturemay be compared to the release of the API in the absence of theadmixture (i.e., the release of the individual API component and/or therelease of the API from the second composition when the secondcomposition is not in admixture with the first composition).

“Modulate,” “modulating,” “modulation,” and grammatical variationsthereof as used herein refer to an increase or reduction in a property(e.g., the pH and/or release of an API) in an admixture of the presentinvention compared to the property in the absence of the admixture. Asused herein, the terms “increase,” “increases,” “increased,”“increasing” and similar terms indicate an elevation in a property(e.g., the pH and/or release of an API) of at least about 5%, 10%, 25%,50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more compared to theproperty in the absence of the admixture. As used herein, the terms“reduce,” “reduces,” “reduced,” “reduction” and similar terms refer to adecrease in a property (e.g., the pH and/or release of an API) of atleast about 5%, 10%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97% or morecompared to the property in the absence of the admixture.

In some embodiments, an admixture may comprise at least two compositions(i.e., a first composition and a second composition). The firstcomposition may modulate the pH of the second composition and/or therelease of an API present in the second composition or vice versa.Admixtures comprising two compositions are described herein for purposesof illustration, but it is understood that the admixture may comprisemore than two different compositions, such as, but not limited to, 2, 3,4, 5, 6, 7, or more compositions. One or more of the compositionspresent in the admixture may modulate a property of another compositionin the admixture. The property modulated may be the same property or adifferent property. In some embodiments, two or more differentcompositions in an admixture may together modulate a property of anothercomposition in the admixture.

An admixture of the present invention may be formed by direct and/orindirect exposure of at least one component in a first composition to atleast one component in a second composition. For example, an admixturemay be formed by mixing and/or combining the first composition andsecond composition prior to, during, and/or after topical application toa subject. The admixture may comprise a single phase even though it maybe prepared from at least two different compositions. A further exampleof direct exposure of a first composition and second composition to forman admixture may occur by applying one or more layers of the secondcomposition onto a subject and then applying one or more layers of thefirst composition onto a subject or vice versa. Indirect exposure mayoccur by applying a second composition onto a subject and then applyinga first composition onto a subject through a substrate, such as, but notlimited to, a cloth, bandage, gauze, and the like, or vice versa to forman admixture.

In certain embodiments, the admixture may be self-emulsifying. Inparticular embodiments, the self-emulsifying admixture comprises a firstcomposition comprising water and a second composition comprising an oil,amphiphilic agent and/or emulsifying agent. Example emulsifying agentsinclude, but are not limited to, phosphatidyl cholines; lecithin;surfactants such as polyethoxylated compounds including Tween 80polysorbate 20, 21, 40, 60, 61, 65, 81, 85, 120 and otherpolyoxyethylene adducts of sorbitan esters, fatty acids, fatty alcohols,lanolin, lanolin alcohols, castor oil (natural or hydrogenated), oralkylbenzenes; and any combination thereof.

A self-emulsifying admixture may form a spontaneous emulsion (e.g., withthe application of minimal or no mechanical energy) upon combining theat least two compositions of the admixture. In some embodiments, theself-emulsifying admixture may not require and/or need heat in order toform a spontaneous emulsion. In some embodiments, a self-emulsifyingadmixture may emulsify spontaneously via a chemical reaction underminimal or no mechanical and/or external force to form a spontaneousemulsion. For example, the self-emulsifying admixture may be formed by asubject and/or third party by mixing the at least two compositions ofthe admixture with their hands. In some embodiments, the minimalmechanical force may provide sufficient shear to emulsify the at leasttwo compositions of the admixture. In some embodiments, the minimalmechanical force to emulsify the at least two compositions of theadmixture may have a shear rate in a range of about 1 s⁻¹ to about 5,000s⁻¹, such as, for example, about 10 s⁻¹ to about 200 s⁻¹, about 100 s⁻¹to about 1000 s⁻¹, about 500 s⁻¹ to about 3000 s⁻¹, or about 10 s⁻¹ toabout 2500 s⁻¹.

The self-emulsifying admixture upon forming an emulsion may containand/or be a single phase. In some embodiments, the self-emulsifyingadmixture may be a coarse emulsion, a microemulsion or a nanoemulsion.In some embodiments, the self-emulsifying admixture may be anon-separating or continuous emulsion and/or a homogeneous composition.In some embodiments, a self-emulsifying admixture may encapsulate ahydrophobic component in a hydrophilic component. As illustrated in FIG.11, in some embodiments, a self-emulsifying admixture may containdroplets of an oil or a hydrophobic phase with water or a hydrophilicphase surrounding the droplets, and the droplets may encapsulate an API.

In some embodiments, a self-emulsifying admixture may be formed uponcombining a hydrophobic composition and a hydrophilic composition. Insome embodiments, the hydrophilic composition may be a hydrogel. In someembodiments, the hydrophobic composition may be determinative as towhether the composition is a self-emulsifying composition. In someembodiments, the hydrophobic composition may not comprise a componentwith a hydrophilic property, such as, for example, a stronglyhydrophilic polyethylene glycol (e.g., PEG 400). In some embodiments,the hydrophobic composition does not comprise a component with ahydrophilic-lipophilic balance (HLB) value greater than 15.

In some embodiments, the admixture is a continuous emulsion (i.e., anon-separating emulsion). In some embodiments, the admixture may remainas a continuous emulsion and/or may stay together as a single phase forat least 1, 2, 3, 4, 5, 6, or more days, or 1, 2, 3, 4, 5, 6, or moreweeks, or 1, 2, 3, 4, 5, 6, or more months. In some embodiments, theadmixture may be a continuous emulsion for a period of time sufficientto apply the composition to a subject. A composition that separates outinto two or more phases within 1 day of combination of two or more partsof the composition is not considered to be a self-emulsifying admixtureand/or a continuous emulsion.

In some embodiments, a self-emulsifying admixture may have a dropletsize (e.g., diameter) of greater than 100 μm. In some embodiments, theself-emulsifying admixture may form or produce an emulsion that may havea droplet size of about 100 μm or less, such as, but not limited to,about 90 μm, 70 μm, 50 μm, 30 μm or less, or any range and/or individualvalue therein. In some embodiments, the self-emulsifying admixture mayform or produce an emulsion that may have a droplet size of greater than1 μm. In some embodiments self-emulsifying admixture may form or producea nanoemulsion that may have a droplet size of about 400 nm or less,such as, but not limited to, about 300 nm, 200 nm, 100 nm, 50 nm orless, or any range and/or individual value therein. In some embodiments,a self-emulsifying admixture may comprise droplets that aresubstantially uniform in size.

The first composition in the admixture may be configured to modulate therelease of an API present in the second composition such as, but notlimited to, an NO releasing API.

In some embodiments, when an admixture is formed comprising the firstand second compositions, water present in the first composition maycontact the second composition to modulate the release of an API presentin the second composition, such as, but not limited to, an NO releasingAPI. Alternatively or in addition, in some embodiments, the firstcomposition in an admixture may modulate the pH of the secondcomposition in the admixture, thereby modulating the release of an APIpresent in the second composition, such as, but not limited to, an NOreleasing API. In some embodiments, the first composition in anadmixture may be configured to supply water to the second composition inthe admixture and/or configured to modulate the pH of the secondcomposition in the admixture. In some embodiments, an admixture of thepresent invention may increase the solubility of an API (e.g., anNO-releasing API) and/or may increase the bioavailability of an API oran active component of an API (e.g., NO).

The inventors of the present invention surprisingly discovered that anadmixture comprising an emulsion of water (i.e., a hydrophilic phase)and oil (i.e., a hydrophobic phase) could be prepared that providedsufficient NO release. It was further surprisingly discovered by theinventors that an admixture having a single phase could be prepared uponcombination of a hydrophobic composition and hydrophilic composition,and that such composition could provide sufficient NO release.Self-emulsifying admixtures as described herein were also surprisinglydiscovered by the inventors.

An admixture of the present invention may provide a particular releasepattern for an API present in the admixture. The API release pattern maybe determined by comparing the amount or concentration of API releasedover a period of time and/or the rate of release of an API from theadmixture over a period of time. In some embodiments, the at least twodifferent compositions present in the admixture are selected to providea particular API release pattern. The API release pattern may bedesirable for a particular injury, disease, disorder, or treatmentindication. In some embodiments, the admixture may be configured toprovide a particular release pattern of an API present in the admixture.

In some embodiments, the at least two different compositions present inthe admixture may be selected to provide the admixture with a pH of lessthan about pH 11, such as, but not limited to, about 11, 10, 9, 8, 7, 6,5, 4, 3, or less. In some embodiments, the at least two differentcompositions present in the admixture may be selected to provide theadmixture with a pH of greater than about pH 4, such as, but not limitedto, about 4, 5, 6, 7, 8, 9, 10, 11, or more. In certain embodiments, theadmixture pH may be between about pH 4 to about pH 11, such as, but notlimited to, about pH 4 to about pH 9, about pH 7 to about pH 9, about pH4 to about pH 8, pH 7 to about pH 10, or about pH 5 to about pH 7. Insome embodiments, at least one of the compositions present in theadmixture may maintain the pH of the admixture in a particular pH range.The pH of the admixture may vary over time and this may cause therelease rate of the API from the admixture to vary over time. Foradmixtures where the pH changes over time, the pH of the admixture maybe measured within about 30 minutes after combination, in someembodiments, within about 10 minutes after combination, and in someembodiments, 2 minutes after combination. In some embodiments the pH ofthe admixture may be measured at about 5 minutes, 30 minutes, 1 hour,and/or 24 hours after combination.

An admixture of the present invention may provide for immediate releaseof the API from the admixture and/or sustained release of the API fromthe admixture. As used herein, immediate release refers to the releaseof 50% or more of the API within 4 hours of mixing and sustained releaserefers to the release of less than 50% of the API within 4 hours ofmixing. In some embodiments, an admixture of the present invention mayincrease the amount of API released and/or the potency of an API presentin at least one composition in the admixture by maintaining the pH ofthe admixture in a particular pH range compared to the release and/orpotency of the API in the composition in the absence of the admixture.In certain embodiments the pH of the admixture is maintained below pH 9.

The API present in the admixture may be released substantiallycontinuously from the admixture over a period of time. “Substantiallycontinuously,” and grammatical variants thereof as used herein refer toa release of an API from the admixture for all or part of the time suchthat on average the release of the API confers an overall beneficialeffect on the subject. Thus, there may be one or more short,intermittent and/or regular time periods in which the API is not beingreleased, but the overall beneficial effect of the API on the subjectremains. In some embodiments, the admixture may provide an API releasepattern that is substantially continuous over a period of time andprovide a therapeutically effective amount of the API over the period oftime. In some embodiments, the amount of API released and/or the APIrelease rate may vary over a period of time. In certain embodiments, theadmixture may comprise two or more (e.g., 2, 3, 4, 5 or more) releaserates for the API.

The admixture may provide an API release pattern that is substantiallyconstant over a period of time. “Substantially constant” as used hereinrefers to a measureable value, such as the amount of API or the APIrelease rate, on average, varying less than about 20%, 15%, 10%, 5%, 1%or less over a period of time. In some embodiments, the API release ratemay be substantially constant for a period of time and vary over anotherconsecutive or nonconsecutive period of time and vice versa.

In some embodiments, the admixture may provide an API release patternhaving a rapid release portion and a substantially constant releaseportion. The rapid release portion may comprise the amount of APIreleased from administration (i.e., t=0) to 2 hours after administrationor any range therein, such as, but not limited to, 0 to 1 hour or 0 to30 minutes after administration. The substantially constant releaseportion may comprise the amount of API released from immediately afterthe rapid release portion to the final amount of API is released. An APImay be released from an admixture of the present invention for anyperiod of time. In some embodiments, an API may be released from theadmixture for at least about 4 hours, 6 hours, 12 hours, 24 hours, 2days, 3, days, 4 days, 5, days, 6 days, 7 days, or more, or any rangeand/or individual value therein. The API released from the admixture maybe released in an amount that overall provides a beneficial effect onthe subject and/or provides a therapeutically effective amount of theAPI over the period of time.

In some embodiments, a greater amount or concentration of the API may bereleased during the rapid release portion compared to the substantiallyconstant release portion or vice versa. In some embodiments, the amountof API released from the admixture during the rapid release portion maybe about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%,300%, 400%, 500%, or more, or any range and/or individual value therein,than the amount of API released during the substantially constantrelease portion. In other embodiments, the amount of API released fromthe admixture during the substantially constant release portion may beabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%,300%, 400%, 500%, or more, or any range and/or individual value therein,than the amount of API released during the rapid release portion.

In particular embodiments, a first composition in an admixture maymodulate the pH of a second composition in the admixture such that whenthe admixture is formed and/or applied to the skin of a subject, the pHof the admixture is less than about 9, in further embodiments, less thanabout 8.5, in still further embodiments, less than about 7, and in yetfurther embodiments, between about 5 and about 8. In some embodiments, afirst composition in an admixture may be configured to maintain and/orstabilize the pH of the admixture in a desired pH range, such as, butnot limited to, a pH range of about 3 to about 11, about 3 to about 9,about 4 to about 7, or about 5 to about 8.

An admixture of the present invention may be suitable for topicaladministration. The admixture may comprise a single phase even though itmay be prepared or formed from two or more different compositions. Theadmixture may be buffered. In some embodiments, the admixture maycomprise a hydrophobic composition and a hydrophilic composition. Incertain embodiments, a hydrophobic composition and/or a hydrophiliccomposition may be a single agent or compound (I.e., component). Inother embodiments, a hydrophobic composition and/or a hydrophiliccomposition may comprise a composition having two or more agents orcompounds. In some embodiments, an admixture may comprise a hydrogel andan ointment. The hydrogel and ointment may form an admixture having asingle phase that is optionally buffered. In some embodiments, theadmixture comprises a hydrogel and an ointment, and the admixture may bein the form of a cream. In some embodiments, the admixture may be aself-emulsifying admixture and may comprise a hydrogel and an ointment.

In certain embodiments, an admixture of the present invention comprisesa hydrophilic composition. The hydrophilic composition comprises atleast one hydrophilic component. The hydrophilic composition may be asolution, suspension, lotion, gel, cream, hydrogel, and the like. Insome embodiments, the hydrophilic composition is in the form of ahydrogel. “Hydrogel,” as used herein, refers to a hydrophilic gelcomprising a gel matrix and water. Water may be present in a hydrophiliccomposition in an amount of about 50% to about 99% by weight of thehydrophilic composition, or any range and/or individual value therein,such as, but not limited to, about 70% to about 99%/o or about 80% toabout 85% by weight of the hydrophilic composition.

The hydrophilic composition may comprise means for maintaining and/orstabilizing the pH of an admixture of the present invention. The meansfor maintaining and/or stabilizing the pH of an admixture may beconfigured to control the pH of the admixture within a desired pH range.Example means for maintaining and/or stabilizing the pH of an admixtureinclude, but are not limited to, buffers. Examples of buffers that maybe used in a hydrophilic composition include, but are not limited to,acetic acid/acetate buffers; hydrochloric acid/citrate buffers;citro-phosphate buffers; phosphate buffers; citric acid/citrate buffers;lactic acid buffers; tartaric acid buffers; malic acid buffers;glycine/HCl buffers; saline buffers such as phosphate buffered saline(PBS), Tris-buffered saline (TBS), Tris-HCl, NaCl, Tween buffered saline(TNT), phosphate buffered saline, Triton X-100 (PBT) and mixturesthereof; cacodylate buffers; barbital buffers; tris buffers; and anycombination thereof.

A buffer may be present in the hydrophilic composition at aconcentration of about 5 mmol to about 2 moles or any range and/orindividual value therein, such as, but not limited to about 10 mmol toabout 1 mole, about 100 mmol to about 750 mmol, or about 200 mmol toabout 500 mmol. In some embodiments, a buffer may be present in thehydrophilic composition in an amount of about 0.1% to about 20% byweight of the hydrophilic composition or any range and/or individualvalue therein, such as, but not limited to, about 0.1% to about 10%,about 1% to about 15%, or about 1% to about 5% by weight of thehydrophilic composition.

In some embodiments, the hydrophilic composition may comprise aphosphate buffer. Example phosphate buffers may include at least onephosphate salt such as, but not limited to, sodium phosphate (e.g.,sodium dihydrogen phosphate, disodium hydrogen phosphate, trisodiumphosphate and sodium aluminum phosphate), potassium phosphate (e.g.,potassium phosphate monobasic and potassium phosphate dibasic), rubidiumphosphate, caesium phosphate, and ammonium phosphate, and/or at leastone phosphoric acid such as, but not limited to, pyrophosphoric acid,triphosphoric acid, and orthophosphoric acid. The hydrophiliccomposition may have a total phosphate concentration of about 5 mmol toabout 1 mole of phosphate or any range and/or individual value therein,such as, but not limited to, about 10 mmol to about 750 mmol, about 150mmol to about 500 mmol, or about 200 mmol to about 400 mmol. In certainembodiments, the hydrophilic composition may have a phosphate bufferpresent in an amount of about 1% to about 20% by weight of thehydrophilic composition, such as, but not limited to about 1% to about15% by weight, about 5% to about 15% by weight, about 5% to about 10% byweight, or about 4% to about 8% by weight of the hydrophiliccomposition.

In certain embodiments, a hydrophilic composition may comprise abuffering agent. Example buffering agents include, but are not limitedto, citric acid, acetic acid, lactic acid, boric acid, succinic acid,malic acid, sodium hydroxide, potassium hydroxide, and any combinationthereof. A buffering agent may be present in a hydrophilic compositionof the present invention in an amount of about 0.01% to about 5% byweight of the hydrophilic composition or any range and/or individualvalue therein, such as, but not limited to, about 0.05% to about 3%,about 1% to about 4%, or about 1.5% to about 3.5% by weight of thehydrophilic composition.

Thus, in certain embodiments, an admixture of the present invention maycomprise a hydrophilic composition such as, but not limited to, ahydrogel, that is optionally buffered. The hydrophilic composition maybe pH dependent. The hydrophilic composition may be configured to have abuffer capacity of at least about 4 to about 8, or any range and/orindividual value therein, such as, but not limited to, about 4 to about7, about 5 to about 6, about 5 to about 8, or about 6 to about 8. Thehydrophilic composition may be configured to maintain and/or stabilizethe pH of an admixture within about 0.5 or more pH units such as, forexample, about 1, 2, or 3 pH units, of the pH of the hydrophiliccomposition. The pH of the admixture may be maintained and/or stabilizedwhen the admixture is formed and/or at the site of application (e.g.,the skin of a subject and/or a wound bed) for the admixture. Forexample, when an admixture comprising a hydrophilic composition having apH of about 5 is formed with an additional composition and applied tothe skin of a subject, the hydrophilic composition may be configured tomaintain and/or stabilize the pH of the admixture within about 0.5 pHunits of the hydrophilic composition pH (i.e., the hydrophiliccomposition may maintain the pH of the admixture in a pH range of about4.5 to 5.5). In some embodiments, a hydrophilic composition may beconfigured to maintain and/or stabilize the pH of an admixture in a pHrange of about pH 3 to about pH 6, about pH 3 to about pH 5, about pH 3to about pH 4, about pH 4 to about pH 8, about pH 4 to about pH 7, aboutpH 4 to about pH 6, about pH 5 to about pH 7, about pH 5 to about pH 6,about pH 6 to about pH 7, or any other range therein. The admixture maythus provide a particular pH to the site of application (e.g., woundbed), which may increase or decrease the pH of the site of applicationin the absence of the admixture.

A hydrophilic composition of the present invention may have any suitablepH, such as a pH of about 1 or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, or 14). In some embodiments, the hydrophilic compositionmay be configured to have a pH in a range of about 3 to about 8 or anyrange and/or individual value therein, such as about 3 to about 4 orabout 4 to about 6. In certain embodiments, the hydrophilic compositionmay be configured to have a pH of about 5. In some embodiments, thehydrophilic composition may be buffered.

A hydrophilic composition may comprise a natural and/or syntheticpolymer. Example polymers include, but are not limited to,polysaccharides such as chitosan and chitin; charged celluloses andpharmaceutically acceptable salts thereof; acrylic acids such aspolyacrylic polymers such as polyacrylic acid, polyacrylate polymers,cross-linked polyacrylate polymers, cross-linked polyacrylic acids,polyacrylic acid polymers commercially available from Lubrizol ofWickliffe, Ohio under the trademark CARBOPOLm, and mixtures thereof; andany combination thereof. In some embodiments, a hydrophilic compositioncomprises a charged cellulose or a pharmaceutically acceptable saltthereof. Example charged celluloses or pharmaceutically acceptable saltsthereof include, but are not limited to, ionic celluloses, carboxymethylcellulose and its salts, hydroxyethyl carboxymethyl cellulose,hydroxypropyl carboxymethyl cellulose, sulfoethyl cellulose,hydroxyethyl sulfoethyl cellulose, hydroxypropyl sulfoethyl cellulose,hydroxyethyl cellulose ethoxylate, hydroxypropylmethyl cellulose, methylcellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxyethylmethylcellulose, carrageenan, chitosan, xanthan gum, sodium alginate,propylene glycol aginate, alginic acid and its salts, and anycombination thereof. In some embodiments, a hydrophilic composition maycomprise hydroxyethyl cellulose ethoxylate, quaternized. In someembodiments, a hydrophilic composition may comprise chitosan.

A polymer, such as, but not limited to, charged cellulose or apharmaceutically acceptable salt thereof, may be present in ahydrophilic composition in an amount of about 0.1% to about 15% byweight of the hydrophilic composition or any range and/or individualvalue therein, such as, but not limited to, about 0.3% to about 10%,about 1% to about 10% or about 1% to about 5% by weight of thehydrophilic composition. In certain embodiments, a polymer, such as, butnot limited to, charged cellulose or a pharmaceutically acceptable saltthereof, may be present in a hydrophilic composition in an amount ofabout 0.1%, 0.3%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, or 15% by weight of the hydrophilic composition or anyrange and/or individual value therein.

A hydrophilic composition may comprise a polyhydric alcohol. Examplepolyhydric alcohols include, but are not limited to, glycerol, glycols,propylene glycol, hexylene glycol, polyethylene glycol, polypropyleneglycol, triethylene glycol, neopental glycols, triethanolamine,diethanolamine, ethanolamione, butylene glycol, polyethylene glycol,n-methyl diethanolamine, isopropanolamine, sorbitol, arabitol,erythritol, HSH, isomalt, lactitol maltitol, mannitol, xylitol,threitol, ribitol, galactitol, fucitol, iditol, inositol, volemitol, andany combination thereof. In some embodiments, a hydrophilic compositionmay comprise a glycol, such as hexylene glycol. A polyhydric alcohol maybe present in a hydrophilic composition in an amount of about 1% toabout 30% by weight of the hydrophilic composition or any range and/orindividual value therein, such as, but not limited to, about 1% to about25%, about 5% to about 15%, about 10% to about 30%, or about 15% toabout 25% by weight of the hydrophilic composition. In certainembodiments, a polyhydric alcohol may be present in a hydrophiliccomposition in an amount of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, or 30% by weight of the hydrophiliccomposition or any range and/or individual value therein.

A hydrophilic composition may comprise a preservative. A preservativemay be present in a hydrophilic composition in an amount of about 0.01%to about 2% by weight of the composition or any range and/or individualvalue therein, such as, but not limited to, about 0.05% to about 1% orabout 0.1% to about 1% by weight of the hydrophilic composition. Incertain embodiments, a preservative is present in a hydrophiliccomposition in an amount of about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%,0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%,0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or2% by weight of the hydrophilic composition or any range and/orindividual value therein. Example preservatives that may be present in ahydrophilic composition of the present invention include, but are notlimited to, sorbic acid, benzoic acid, methyl-paraben, propyl-paraben,methylchloroisothiazolinone, metholisothiazolinone, diazolidinyl urea,chlorobutanol, triclosan, benzethonium chloride, p-hydroxybenzoate,chlorhexidine, digluconate, hexadecyltrimethyl ammonium bromide,alcohols, benzalkonium chloride, boric acid, bronopol, butylparaben,butylene calcium acetate, calcium chloride, calcium lactate, carbondioxide, cationic, and bentonite, cetrimide, cetylpyridinium chloride,chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, citric acidmonohydrate, cresol, dimethyl ether, ethylparaben, glycerin, hexetidine,imidurea, isopropyl alcohol, lactic acid, monothioglycerol, penteticacid, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuricacetate, phenylmercuric borate, phenylmercuric nitrate, potassiumbenzoate, potassium metabisulfite, potassium sorbate, propionic acid,propyl gallate, propylene glycol, sodium acetate, sodium benzoate,sodium borate, sodium lactate, sodium sulfite, sodium propionate, sodiummetabisulfite, xylitol, sulphur dioxide, carbon dioxide, and anycombination thereof.

A hydrophilic composition may comprise a neutralizing agent. Aneutralizing agent may be present in a hydrophilic composition in anamount sufficient to provide the hydrophilic composition with a pH ofabout 3 to about 8, or any range and/or individual value therein, suchas, but not limited to, about 4 to about 7 or about 6 to about 7. Insome embodiments, a neutralizing agent adjusts the pH of the hydrophiliccomposition. In certain embodiments of the present invention, aneutralizing agent may be present in a hydrophilic composition of thepresent invention in an amount sufficient for the hydrophiliccomposition to have a pH of about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,7.5, or 8 or any range and/or individual value therein. Exampleneutralizing agents that may be present in a hydrophilic compositioninclude, but are not limited to, bases such as sodium hydroxide,potassium hydroxide, and mixtures thereof; acids such as hydrochloricacid, citric acid, acetic acid, and mixtures thereof; sodium carbonate;trolamine; tromethamine; aminomethyl propanol; triisopropanolamine;aminomethyl propanol; tetrahydroxypropyl ethylenediamine; tetrasodiumEDTA; suttocide A; and any combination thereof.

According to some embodiments, a hydrophilic composition may beantimicrobial. A hydrophilic composition may be cosmetically elegant.“Cosmetically elegant” as used herein, refers to a composition that isattractive for application to the skin, which may include mucosa. Insome embodiments, a composition may be cosmetically elegant for the skinand/or mucosa. A cosmetically elegant composition of the presentinvention may have one or more of the following properties: suitableconsistency or viscosity for topical application (e.g., easy to spreadonto the skin and does not run), suitable texture for topicalapplication (e.g., a smooth or soft composition that is not gritty),ability to absorb and/or permeate the skin, non-sticky or not tacky,does not leave a residue, leaves the skin feeling good, and afterapplication does not leave the skin oily or dry. In some embodiments, ahydrophilic composition may have a viscosity of about 5,000 cP(centipoise) to about 100,000 cP, or any range and/or individual valuetherein, such as, but not limited to, about 10,000 cP to about 50,000cP, about 20,000 cP to about 40,000 cP, about 30,000 cP to about 50,000cP, about 50,000 cP to about 100,000 cP, or about 30,000 cP to about75,000 cP.

A hydrophilic composition such as, but not limited to, a hydrogel, ofthe present invention may be suitable in an admixture of the presentinvention with one or more, such as, but not limited to, 2, 3, 4, ormore, different compositions. A hydrophilic composition, such as, butnot limited to, a hydrogel, of the present invention may be used as adrug delivery system and/or a drug release system when in an admixtureof the present invention. For example, a hydrophilic composition may beconfigured to modulate the release of an active pharmaceuticalingredient (API) in a second composition when an admixture comprisingthe hydrophilic composition and second composition is formed and/oradministered. Alternatively or in addition, a hydrophilic compositionmay be configured to modulate the pH of a second composition when anadmixture comprising the hydrophilic composition and second compositionis formed and/or administered. In some embodiments, a hydrophiliccomposition may be configured to modulate the pH of a second compositioncomprising a nitric oxide (NO) releasing API and/or the rate of NOrelease from a NO releasing API when an admixture comprising thehydrophilic composition and second composition is formed and/oradministered. In certain embodiments, the second composition may be ahydrophobic composition, such as, but not limited to, an ointment. Insome embodiments, a hydrophilic composition may be configured tomodulate the pH of an admixture in which it is present within a desiredpH range.

An admixture of the present invention may have any suitable pH. In someembodiments, the admixture may have a pH of about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, or 14. In some embodiments, the admixture may beconfigured to have a pH in a range of about pH 2 to about pH 9, such asabout pH 4 to about pH 9, about pH 3 to about pH 6, about pH 3 to aboutpH 5, about pH 3 to about pH 4, about pH 4 to about pH 8, about pH 4 toabout pH 7, about pH 4 to about pH 6, about pH 5 to about pH 7, about pH5 to about pH 6, about pH 6 to about pH 7, or about pH 5 to about pH 8,or any other range therein. In certain embodiments, an admixture of thepresent invention is buffered to a suitable pH.

An admixture of the present invention may comprise an activepharmaceutical ingredient (API). Any suitable API or combinations ofAPIs may be included in an admixture of the present invention. Examplesof APIs include, but are not limited to, antimicrobial agents, anti-acneagents, anti-inflammatory agents, analgesic agents, anesthetic agents,antihistamine agents, antiseptic agents, immunosuppressants,antihemorrhagic agents, vasodilators, wound healing agents, anti-biofilmagents, and any combination thereof. Example APIs include, but are notlimited to, those described in International Application No.PCT/US2013/028223, which is incorporated herein by reference in itsentirety. In some embodiments, the admixture and/or API does notcomprise acidified nitrite. “Acidified nitrite”, as used herein, refersto a nitric oxide releasing composition where the primary mechanism ofnitric oxide release is when a nitrite is reduced, in the presence of anacid, to dinitrogen trioxide, which can dissociate into nitric oxide andnitrous oxide.

Examples of antimicrobial agents include, but are not limited to,penicillins and related drugs, carbapenems, cephalosporins and relateddrugs, erythromycin, aminoglycosides, bacitracin, gramicidin, mupirocin,chloramphenicol, thiamphenicol, fusidate sodium, lincomycin,clindamycin, macrolides, novobiocin, polymyxins, rifamycins,spectinomysin, tetracyclines, vanomycin, teicoplanin, streptogramins,anti-folate agents including sulfonamides, trimethoprim and itscombinations and pyrimethamine, synthetic anti-bacterials includingnitrofurans, methenamine mandelate and methenamine hippurate,nitroimidazoles, quinolones, fluoroquinolones, isoniazid, ethambutol,pyrazinamide, para-aminosalicylic acid (PAS), cycloserine, capreomycin,ethionamide, prothionamide, thiacetazone, viomycin, eveminomycin,glycopeptide, glyclyclycline, ketolides, oxazolidinone; imipenen,amikacin, netilmicin, fosfomycin, gentamycin, ceftriaxone, Ziracin,Linezolid, Synercid, Aztreonam, and Metronidazole, Epiroprim,Sanfetrinem sodium, Biapenem, Dynemicin, Cefluprenam, Cefoselis,Sanfetrinem celexetil, Cefpirome, Mersacidin, Rifalazil, Kosan,Lenapenem, Veneprim, Sulopenem, ritipenam acoxyl, Cyclothialidine,micacocidin A, carumonam, Cefozopran and Cefetamet pivoxil.

Examples of topical anti-acne agents include, but are not limited to,adapalene, azelaic acid, benzoyl peroxide, clindamycin and clindamycinphosphate, doxycycline, erythromycin, keratolytics such as salicylicacid and retinoic acid (Retin-A″), norgestimate, organic peroxides,retinoids such as isotretinoin and tretinoin, sulfacetamide sodium, andtazarotene. Particular anti-acne agents include adapalene, azelaic acid,benzoyl peroxide, clindamycin (e.g., clindamycin phosphate), doxycycline(e.g., doxycycline hyclate), erythromycin, isotretinoin, norgestimate,sulfacetamide sodium, tazarotene, etretinate and acetretin.

Examples of antihistamine agents include, but are not limited to,diphenhydramine hydrochloride, diphenhydramine salicylate,diphenhydramine, chlorpheniramine hydrochloride, chlorpheniraminemaleate isothipendyl hydrochloride, tripelennamine hydrochloride,promethazine hydrochloride, methdilazine hydrochloride, and the like.Examples of local anesthetic agents include dibucaine hydrochloride,dibucaine, lidocaine hydrochloride, lidocaine, benzocaine,p-buthylaminobenzoic acid 2-(die-ethylamino) ethyl ester hydrochloride,procaine hydrochloride, tetracaine, tetracaine hydrochloride,chloroprocaine hydrochloride, oxyprocaine hydrochloride, mepivacaine,cocaine hydrochloride, piperocaine hydrochloride, dyclonine anddyclonine hydrochloride.

Examples of antiseptic agents include, but are not limited to, alcohols,quaternary ammonium compounds, boric acid, chlorhexidine andchlorhexidine derivatives, iodine, phenols, terpenes, bactericides,disinfectants including thimerosal, phenol, thymol, benzalkoniumchloride, benzethonium chloride, chlorhexidine, povidone iode,cetylpyridinium chloride, eugenol and trimethylammonium bromide.

Examples of anti-inflammatory agents include, but are not limited to,nonsteroidal anti-inflammatory agents (NSAIDs); propionic acidderivatives such as ibuprofen and naproxen; acetic acid derivatives suchas indomethacin; enolic acid derivatives such as meloxicam,acetaminophen; methyl salicylate; monoglycol salicylate; aspirin;mefenamic acid; flufenamic acid; indomethacin; diclofenac; alclofenac;diclofenac sodium; ibuprofen; ketoprofen; naproxen; pranoprofen;fenoprofen; sulindac; fenclofenac; clidanac; flurbiprofen; fentiazac;bufexamac; piroxicam; phenylbutazone; oxyphenbutazone; clofezone;pentazocine; mepirizole; tiaramide hydrochloride; steroids such asclobetasol propionate, bethamethasone dipropionate, halbetasolproprionate, diflorasone diacetate, fluocinonide, halcinonide,amcinonide, desoximetasone, triamcinolone acetonide, mometasone furoate,fluticasone proprionate, betamethasone diproprionate, triamcinoloneacetonide, fluticasone propionate, desonide, fluocinolone acetonide,hydrocortisone vlaerate, prednicarbate, triamcinolone acetonide,fluocinolone acetonide, hydrocortisone and others known in the art,predonisolone, dexamethasone, fluocinolone acetonide, hydrocortisoneacetate, predonisolone acetate, methylpredonisolone, dexamethasoneacetate, betamethasone, betamethasone valerate, flumetasone,fluorometholone, beclomethasone diproprionate, fluocinonide, topicalcorticosteroids, and may be one of the lower potency corticosteroidssuch as hydrocortisone, hydrocortisone-21-monoesters (e.g.,hydrocortisone-21-acetate, hydrocortisone-21-butyrate,hydrocortisone-21-propionate, hydrocortisone-21-valerate, etc.),hydrocortisone-17,21-diesters (e.g., hydrocortisone-17,21-diacetate,hydrocortisone-17-acetate-21-butyrate, hydrocortisone-17,21-dibutyrate,etc.), alclometasone, dexamethasone, flumethasone, prednisolone, ormethylprednisolone, or may be a higher potency corticosteroid such asclobetasol propionate, betamethasone benzoate, betamethasonedipropionate, diflorasone diacetate, fluocinonide, mometasone furoate,triamcinolone acetonide.

Examples of analgesic agents include, but are not limited to,alfentanil, benzocaine, buprenorphine, butorphanol, butamben, capsaicin,clonidine, codeine, dibucaine, enkephalin, fentanyl, hydrocodone,hydromorphone, indomethacin, lidocaine, levorphanol, meperidine,methadone, morphine, nicomorphine, opium, oxybuprocaine, oxycodone,oxymorphone, pentazocine, pramoxine, proparacaine, propoxyphene,proxymetacaine, sufentanil, tetracaine and tramadol.

Examples of anesthetic agents include, but are not limited to, alcoholssuch as phenol; benzyl benzoate; calamine; chloroxylenol; dyclonine;ketamine; menthol; pramoxine; resorcinol; troclosan; procaine drugs suchas benzocaine, bupivacaine, chloroprocaine; cinchocaine; cocaine;dexivacaine; diamocaine; dibucaine; etidocaine; hexylcaine;levobupivacaine; lidocaine; mepivacaine; oxethazaine; prilocaine;procaine; proparacaine; propoxycaine; pyrrocaine; risocaine; rodocaine;ropivacaine; tetracaine; and derivatives, such as pharmaceuticallyacceptable salts and esters including bupivacaine HCl, chloroprocaineHCl, diamocaine cyclamate, dibucaine HCl, dyclonine HCl, etidocaine HCl,levobupivacaine HCl, lidocaine HCl, mepivacaine HCl, pramoxine HCl,prilocaine HCl, procaine HCl, proparacaine HCl, propoxycaine HCl,ropivacaine HCl, and tetracaine HCl.

Examples of antihemorrhagic agents include, but are not limited to,thrombin, phytonadione, protamine sulfate, aminocaproic acid, tranexamicacid, carbazochrome, carbaxochrome sodium sulfanate, rutin andhesperidin.

An API may be present in any one of the compositions used to form anadmixture of the present invention. In certain embodiments, at least onecomposition used to form an admixture comprises a nitric oxide(NO)-releasing API. In some embodiments, at least composition used toform an admixture does not contain an API, such as, but not limited to,a NO-releasing API. In some embodiments, a composition used to form anadmixture may comprise at least one API, but the composition does notcomprise a NO-releasing API. In certain embodiments, an admixturecomprises a hydrophilic composition and the hydrophilic composition doesnot comprise a NO-releasing API.

In certain embodiments, an admixture of the present invention maycomprise at least one API, such as, but not limited to, a nitricoxide-releasing active pharmaceutical ingredient. In some embodiments,an admixture of the present invention comprises a nitric oxide-releasingactive pharmaceutical ingredient in an amount of about 0.01% to about 5%w/w of nitric oxide or any range and/or individual value therein, suchas about 0.1% to about 3%, about 0.1% to about 1.5%, or about 1% toabout 5% w/w of nitric oxide.

“Nitric oxide releasing active pharmaceutical ingredient” and “NOreleasing API,” as used herein, refer to a compound or other compositionthat provides nitric oxide to the skin of a subject, but is not gaseousnitric oxide. In some embodiments, the NO releasing API includes anitric oxide-releasing compound, hereinafter referred to as a“NO-releasing compound.” A NO-releasing compound includes at least oneNO donor, which is a functional group that may release nitric oxideunder certain conditions. In some embodiments, the at least one NO donorof a NO-releasing compound releases NO when in contact with acomposition of the present invention. In certain embodiments, acomposition of the present invention modulates the amount of NO releasedfrom a NO-releasing compound and/or the rate of NO released from aNO-releasing compound. In some embodiments, a composition of the presentinvention increases the amount of NO released from a NO-releasingcompound and/or the rate of NO released from a NO-releasing compound.

Any suitable NO-releasing compound may be used. In some embodiments, theNO-releasing compound includes a small molecule compound that includes aNO donor group. “Small molecule compound” as used herein is defined as acompound having a molecular weight of less than 500 daltons, andincludes organic and/or inorganic small molecule compounds. In someembodiments, the NO-releasing compound includes a macromolecule thatincludes a NO donor group. A “macromolecule” is defined herein as anycompound that has a molecular weight of 500 daltons or greater. Anysuitable macromolecule may be used, including crosslinked ornon-crosslinked polymers, dendrimers, metallic compounds, organometalliccompounds, inorganic-based compounds, and other macromolecularscaffolds. In some embodiments, the macromolecule has a nominal diameterranging from about 0.1 nm to about 100 μm and may comprise theaggregation of two or more macromolecules, whereby the macromolecularstructure is further modified with an NO donor group.

In some embodiments, the NO-releasing compound includes adiazeniumdiolate functional group as a NO donor. The diazeniumdiolatefunctional group may produce nitric oxide under certain conditions, suchas upon exposure to water. As another example, in some embodiments, theNO-releasing compound includes a nitrosothiol functional group as the NOdonor. The NO donor may produce nitric oxide under certain conditions,such as upon exposure to light. Examples of other NO donor groupsinclude nitrosamine, hydroxyl nitrosamine, hydroxyl amine andhydroxyurea. Any suitable combination of NO donors and/or NO-releasingcompounds may also be used in a second composition as described herein.Additionally, the NO donor may be incorporated into or onto the smallmolecule or macromolecule through covalent and/or non-covalentinteractions.

A NO-releasing macromolecule may be in the form of a NO-releasingparticle, such as those described in U.S. Application Publication No.2009/0214618 and U.S. Pat. No. 8,282,967, the disclosures of which areincorporated by reference herein in their entirety. Other non-limitingexamples of NO-releasing compounds include NO-releasing zeolites asdescribed in United States Patent Publication Nos. 2006/0269620 or2010/0331968; NO-releasing metal organic frameworks (MOFs) as describedin United States Patent Application Publication Nos. 2010/0239512 or2011/0052650; NO-releasing multi-donor compounds as described inInternational Publication No. WO/2013/029009; NO-releasing dendrimers ormetal structures as described in U.S. Publication No. 2009/0214618;nitric oxide releasing coatings as described in U.S. Publication No.2011/0086234; and compounds as described in U.S. Publication No.2010/0098733. The disclosures of each of the references in thisparagraph are incorporated herein by reference in their entirety.Additionally, NO-releasing macromolecules may be fabricated as describedin International Publication No. WO/2012/100174, the disclosure of whichis incorporated herein by reference in its entirety.

In some embodiments, an admixture of the present invention may increasethe amount of NO released from the admixture compared to the amount ofNO released from at least one composition used to form the admixtureover the same period of time. For example, when an admixture comprisinga hydrophilic composition and a hydrophobic composition such as, but notlimited to, an ointment as described herein, is formed, the amount of NOreleased from the admixture may be increased compared to the amount ofNO released from the hydrophobic composition alone (i.e., in the absenceof the hydrophilic composition or admixture). In certain embodiments, anadmixture of the present invention may increase the amount of NOreleased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90,100%, 150%, 200%, 300%, 400%, or more, or any range and/or individualvalue therein, compared to the amount of NO released from at least onecomposition used to form the admixture over the same period of time. Anadmixture of the present invention may release about 1.5 to about 100times more NO than the amount of NO from at least one composition usedto form the admixture over the same period of time, or any range and/orindividual value therein, such as, but not limited to about 2 to about10 times more NO or about 5 to about 50 times more NO.

According to some embodiments, an admixture comprises means forstabilizing and/or maintaining the pH of the admixture. Example meansfor stabilizing and/or maintaining the pH of the admixture include, butare not limited to, buffers such as those described herein. In someembodiments, the admixture may comprise a nitric oxide-releasing activepharmaceutical ingredient in an amount of about 0.01% to about 5% w/w ofnitric oxide and have a pH of about 4 to about 9. An admixture of thepresent invention may be cosmetically elegant and/or antimicrobial. Insome embodiments, an admixture of the present invention isself-emulsifying.

In some embodiments, an admixture of the present invention may comprisea hydrophilic composition as described herein and a hydrophobiccomposition. The hydrophobic composition may be a liquid, solution,ointment, and the like. The hydrophobic composition comprises at leastone hydrophobic component, such as, but not limited to, a hydrophobicbase. Example hydrophobic compositions include those described inInternational Application Nos. PCT/US2010/046173 and PCT/US2013/028223,which are incorporated herein by reference in their entirety. In someembodiments, the hydrophobic composition is an ointment.

In certain embodiments, an admixture of the present invention maycomprise a polymer, such as, but not limited to, a charged cellulose ora pharmaceutically acceptable salt thereof; a polyhydric alcohol; ahydrophobic base; an API; and optionally an amphiphilic compound or anemulsifying agent. In some embodiments, the API may comprise aNO-releasing compound. The admixture may further comprise a buffer, suchas, but not limited to, a phosphate buffer, and be buffered to a pH ofabout 4 to about 9 or any range and/or individual value therein.

At least one hydrophobic base may be present in an admixture of thepresent invention. In some embodiments, a hydrophobic base may bepresent in a hydrophobic composition that may be used to form anadmixture of the present invention. “Hydrophobic base” as used hereinrefers to a natural and/or synthetic fat, wax, oil, and/or the like. Anysuitable hydrophobic base may be used in an admixture of the presentinvention. In certain embodiments, an admixture comprises two or morehydrophobic bases, such as, but not limited to, 2, 3, 4, 5, or morehydrophobic bases. In certain embodiments, a hydrophobic base inaddition to having hydrophobic properties, may also have hydrophilicproperties and thus may be an amphiphilic base. Example hydrophobicbases include, but are not limited to, branched and unbranchedhydrocarbons, branched and unbranched hydrocarbon waxes, vaseline,hydrocarbon gel, liquid paraffin, white petrolatum, petrolatum,microcrystalline wax, andelilla wax, carnauba wax, lanolin (wool wax),wool wax alcohol, esparto grass wax, cork wax, guaruma wax, rice branwax, sugar cane wax, berry wax, ouricury wax, soy wax, jojoba oil,uropygial grease, ceresine, paraffin waxes, micro waxes, plant oils,animal oils, carnauba wax, beeswax, cacao butter, hard fat, mineral oil,vegetable oil, avocado oil, borage oil, canola oil, castor oil,chamomile oil, coconut oil, corn oil, cottonseed oil, rapeseed oil,evening primrose oil, safflower oil, sunflower oil, soybean oil, sweetalmond, palm oil, palm kernel oil, arctium lappa seed oil, sesame oil,borgo officialis seed oil, brassica campestris oleifera oil, brevoortiaoil, bubulum oil, cistus ladaniferus oil, elaeis guineensis oil, almondoil, pine oil, olive oil, peanut oil, wheat germ oil, grape seed oil,thistle oil, lard, tallow, palm olein, illipe butter, shea butter, cocoabutter, kokum butter, sal butter, lecithin, japan wax lanolin, partiallyhydrogenated vegetable oils, hydrophobic polymers, and any combinationthereof.

In some embodiments, a hydrophobic base may comprise a hydrophobicpolymer. Any suitable hydrophobic polymer may be used in an admixture ofthe present invention. Example hydrophobic polymers include, but are notlimited to hydrocarbon polymers and/or co-polymers, aromaticpolyurethanes, silicone rubber, polysiloxanes, polycaprolactone,polycarbonate, polyvinylchloride, polyethylene, polyethylene glycols(6-4000), poly-L-lactide, poly-DL-glycolide, polyetheretherketone(PEEK), polyamide, polyimide and polyvinyl acetate. In certainembodiments, a hydrophobic base may be an amphiphilic base, such as, butnot limited to, a polyethylene glycol (6-4000). In particularembodiments of the present invention, an admixture of the presentinvention comprises one or more hydrocarbon polymers and/or co-polymers.In certain embodiments, an admixture of the present invention maycomprise one or more hydrocarbon polymers and/or co-polymers, such as,but not limited to, those commercially available from Calumet SpecialtyProducts Partners of Indianapolis, Ind. under the trademark Versageleand/or those commercially available from Croda International Plc of EastYorkshire, United Kingdom under the trade name Crodabase SQ.

In some embodiments, an admixture may comprise at least one hydrophobicbase comprising one or more plant and/or mineral oils. Any suitable oilmay be used in the admixtures of the present invention. Example mineraloils include, but are not limited to, light mineral oil, white mineraloil, paraffinic oils, naphtenic oils, aromatic oils, and any combinationthereof.

One or more hydrophobic bases may be present in a hydrophobiccomposition used to form an admixture of the present invention. One ormore hydrophobic bases may be present in a hydrophobic composition at aconcentration from about 2% to about 99% by weight of the hydrophobiccomposition or any range and/or individual value therein, such as, butnot limited to, from about 2% to about 20% by weight, about 35% to about99% by weight, about 35% to about 90% by weight, about 25% to about 50%by weight, about 40% to about 80% by weight, about 65% to about 95% byweight, about 70% to about 80% by weight, or about 50% to about 70% byweight of the hydrophobic composition. In certain embodiments, one ormore hydrophobic bases may be present in a hydrophobic composition usedto form an admixture at a concentration from about 50% to about 70% byweight of the hydrophobic composition.

“Amphiphilic compound” as used herein refers to a compound comprisinghydrophilic and hydrophobic properties. An amphiphilic compound maycomprise two or more compounds, each of which may provide thehydrophilic property and/or the hydrophobic property. In someembodiments, the amphiphilic compound may comprise one compound havinghydrophilic and hydrophobic properties. In particular embodiments of thepresent invention, an amphiphilic compound may absorb moisture withoutsubstantially absorbing vaporous moisture. An amphiphilic compound mayhave a hydrophilic-lipophilic balance (HLB) value of 12 to 20 or anyrange and/or individual value therein, such as, but not limited to, 15to 20 or 18 to 20. In certain embodiments of the present invention, anamphiphilic compound may have a HLB value of 19.

Example amphiphilic compounds include, but are not limited to, fattyacid esters. One or more fatty acid ester(s) may be present in anadmixture of the present invention, such as 2, 3, 4, or more fatty acidesters. Example fatty acid esters include, but are not limited to,C₆-C₂₂ alkyl and/or alkenyl fatty acid esters such as methyl laurate,ethyl laurate, ethyl myristate, ethyl palmitate, ethyl linoleate, propylisobutylate, isopropyl laurate, isopropyl myristate, isopropylpalmitate, oleyl myristate, oleyl stearate, and oleyl oleate;ether-esters such as fatty acid esters of ethoxylated fatty alcohols;polyhydric alcohol esters such as ethylene glycol mono- and di-fattyacid esters, diethylene glycol mono- and di-fatty acid esters;polyethylene glycol (6-2000) fatty acid mono- and/or diesters such asPEG-6-laurate, PEG-6-stearate, PEG-8-dilaurate, PEG-8-distearate, etc.;polyethylene glycol glycerol fatty acid esters such as PEG-20-glyceryllaurate, PEG-20-glyceryl stearate, and PEG-20-glyceryl oleate; propyleneglycol mono- and di-fatty acid esters; polypropylene glycol 2000monooleate; polypropylene glycol 2000 monostearate; ethoxylatedpropylene glycol monostearate; glyceryl mono- and di-fatty acid esters;polyglycerol fatty acid esters such as polyglyceryl-10 laurate, etc.;ethoxylated glyceryl monostearate; 1,3-butylene glycol monostearate;1,3-butylene glycol distearate; polyoxyethylene polyol fatty acid ester;sorbitan fatty acid esters including sorbitan trioleate and sorbitanmonolaurate; polyethylene glycol sorbitan fatty acid esters such asPEG-6 sorbitan monooleate; polyoxyethylene sorbitan fatty acid estersincluding polyoxyethylene (20) sorbitan monolaurate; sucrose fatty acidesters such as saccharose monopalmitate and saccharose monostearate; waxesters such as beeswax, spermaceti, myristyl myristate, stearyl stearateand arachidyl behenate; polyethylene glycol alkyl ethers such as PEG-10oleyl ether or PEG-9 cetyl ether, polyethylene glycol alkyl phenols suchas PEG-10-100 nonyl phenol; polyoxyethylene-polyoxypropylene blockcopolymers such as poloxamer 188; sterol esters such as cholesterolfatty acid esters, and any combination thereof.

In certain embodiments, a fatty acid ester may comprise a polyethyleneglycol (PEG) glyceride. The polyethylene glycol portion of a PEGglyceride may provide the hydrophilic property of an amphiphiliccompound and may include, but is not limited to, PEG 5-1000 or any rangeand/or individual value therein, and any combination thereof. Theglyceride portion of a PEG glyceride may provide the hydrophobicproperty of an amphiphilic compound and may include, but is not limitedto, a natural and/or hydrogenated oil, such as but not limited to,castor oil, hydrogenated castor oil, vitamin A, vitamin D, vitamin E,vitamin K, a plant oil (e.g., corn oil, olive oil, peanut oil, palmkernel oil, apricot kernel oil, almond oil, etc.), and any combinationthereof. Example polyethylene glycol (PEG) glycerides include, but arenot limited to, PEG-20 castor oil, PEG-20 hydrogenated castor oil,PEG-20 corn glycerides, PEG-20 almond glycerides; PEG-23 trioleate,PEG-40 palm kernel oil, PEG-8 caprylic/capric glycerides, PEG-6caprylic/capric glycerides, lauroyl macrogol-32 glyceride, stearoylmacrogol glyceride, tocopheryl PEG-1000 succinate, and any combinationthereof. In some embodiments, a fatty acid ester may comprise a PEG 5-30(i.e., PEG 5, 6, 7, 8, 9, 10, etc.) and a caprylic/capric glyceride. Inparticular embodiments, an admixture may comprise aPEG-5-caprylic/capric glyceride, a PEG-6-caprylic/capric glyceride, aPEG-7-caprylic/capric glyceride, and/or a PEG-8-caprylic/capricglyceride. In certain embodiments, an admixture may comprise one or morefatty acid esters such as, but not limited to, those commerciallyavailable from Sasol of Hamburg, Germany under the trademark SOFTIGEN®.

An amphiphilic compound may be present in a hydrophobic composition usedto form an admixture of the present invention at a concentration fromabout 1% to about 30% by weight of the hydrophobic composition or anyrange and/or individual value therein, such as, but not limited to, fromabout 2% to about 20% by weight, about 1% to about 10% by weight, about1% to about 5% by weight, or about 5% to about 15% by weight of thehydrophobic composition. In certain embodiments, an amphiphilic compoundis present in a hydrophobic composition used to form an admixture of thepresent invention at a concentration of about 10% by weight of thehydrophobic composition.

An admixture of the present invention may further comprise one or moreexcipients. In some embodiments, one or more excipients may be presentin a hydrophobic composition that may be used to form an admixture ofthe present invention. Excipients for use in pharmaceutical compositionsare well-known in the art and examples may be found in the Handbook ofPharmaceutical Excipients (Rowe, R. C. et al., APhA Publications; 5thed., 2005). Classes of excipients may include, but are not limited to,an emollient, a humectant, a cosolvent, a pH modifier, a water repellingagent, an anti-foaming agent, a surfactant, a solubilizer, anemulsifying agent, a wetting agent, a penetration enhancer, anantioxidant, and/or a solvent. The excipients may be present in anadmixture of the present invention at any suitable concentration. Insome embodiments, an excipient may be present in a hydrophobiccomposition used to form an admixture of the present invention at aconcentration from about 1% to about 20% by weight of the hydrophobiccomposition or any range and/or individual value therein, such as, butnot limited to, from about 1% to about 15% by weight, about 1% to about10% by weight, or about 5% to about 10% by weight of the hydrophobiccomposition.

In some embodiments, an admixture may further comprise a cosolvent. Acosolvent may be present in a hydrophobic composition used to form anadmixture of the present invention at a concentration from about 1% toabout 30% by weight of the hydrophobic composition or any range and/orindividual value therein, such as, but not limited to, from about 2% toabout 20% by weight, about 5% to about 25% by weight, or about 5% toabout 15% by weight of the hydrophobic composition. In certainembodiments of the present invention, a cosolvent may be present in ahydrophobic composition used to form an admixture of the presentinvention at a concentration from about 10% to about 15% by weight ofthe hydrophobic composition.

Example cosolvents include, but are not limited to, a fatty acid ester,propylene glycol, glycerol, polyethylene glycol, a silicone such ascyclomethicone, and any combination thereof. In some embodiments, acosolvent may comprise a neutral oil. In certain embodiments, acosolvent comprises a caprylic and/or capric fatty acid ester, such as acaprylic and/or capric triglyceride. Example cosolvents include, but arenot limited to, those commercially available from Sasol of Hamburg,Germany under the trademark MIGLYOLL.

An admixture may comprise a humectant. Any suitable humectant orcombination of humectants may be used. A humectant may be present in ahydrophobic composition used to form an admixture of the presentinvention at a concentration from about 1% to about 25% by weight of thehydrophobic composition or any range and/or individual value therein,such as, but not limited to, from about 2% to about 20% by weight, about5% to about 10% by weight, or about 5% to about 15% by weight of thehydrophobic composition. In certain embodiments, a humectant may bepresent in a hydrophobic composition used to form an admixture of thepresent invention at a concentration from about 10% to about 15% byweight of the hydrophobic composition.

Example humectants include, but are not limited to, polyhydric alcohols,such as glycols such as diethylene glycol monoethyl ether andmethoxypolyethyleneglycol; glycerols such as propylene glycol, glycerol,isopropanol, ethanol, ethylene glycol, polyethylene glycol,ethoxydiglycol or mixtures thereof; sugar polyols, such as sorbitol,xylitol and maltitol; polyols such as polydextroses; dimethylisosorbide; quillaia; urea; and any combination thereof. In particularembodiments of the present invention, a humectant comprises an alkyleneglycol, such as hexylene glycol, butylene glycol, pentylene glycol, andany combination thereof.

An admixture may comprise an emulsifying agent. Any suitable emulsifyingagent or combination of emulsifying agents may be used. An emulsifyingagent may be present in a hydrophobic composition used to form anadmixture of the present invention at a concentration from about 2% toabout 97% by weight of the hydrophobic composition or any range and/orindividual value therein, such as, but not limited to, from about 2% toabout 20% by weight, about 5% to about 15% by weight, about 10% to about30%, by weight, about 25% to about 99% by weight, or about 25% to about70% by weight of the hydrophobic composition. In certain embodiments, anemulsifying agent may be present in a hydrophobic composition used toform an admixture of the present invention at a concentration from about10% to about 50% by weight of the hydrophobic composition.

Example emulsifying agents include, but are not limited to, phosphatidylcholines; lecithin; surfactants such as polyethoxylated compoundsincluding tween 80 polysorbate 20, 21, 40, 60, 61, 65, 81, 85, 120 andother polyoxyethylene adducts of sorbitan esters, fatty acids, fattyalcohols, lanolin, lanolin alcohols, castor oil (natural orhydrogenated), or alkylbenzenes; fatty alcohols such as cetyl alcohol,stearyl alcohol, behenyl alcohol, myristyl alcohol, and cetostearylalcohol; fatty acid esters such as those commercially available fromSasol of Hamburg, Germany under the trademark MIGLYOL®; and anycombination thereof.

According to some embodiments, a hydrophobic composition used to form anadmixture of the present invention may comprise at least one hydrophobicbase in an amount of about 55% to about 99% by weight of the hydrophobiccomposition and an API, such as, but not limited to, a nitricoxide-releasing API. In certain embodiments, the at least onehydrophobic base may be present in the hydrophobic composition in anamount of about 70% to about 80% by weight of the hydrophobiccomposition. In some embodiments, an amphiphilic base may be present inthe hydrophobic composition in an amount of about 15% to about 45% byweight of the hydrophobic composition. The hydrophobic composition mayoptionally comprise a cosolvent in an amount of about 2% to about 30% byweight of the hydrophobic composition, a humectant in an amount of about5% to about 10% by weight of the hydrophobic composition, an emulsifyingagent in an amount of about 5% to about 25% by weight of the hydrophobiccomposition, and/or an amphiphilic compound in an amount of about 1% toabout 10% by weight of the hydrophobic composition.

In some embodiments, an admixture of the present invention may comprisea hydrophilic composition, which may comprise water, a polymer such as,but not limited to, a charged cellulose or a pharmaceutically acceptablesalt thereof, and a polyhydric alcohol, in an admixture with ahydrophobic composition, which may comprise at least one hydrophobicbase. The hydrophilic composition may be buffered and/or may be in theform of a hydrogel. The hydrophobic composition may be in the form of anointment. In some embodiments, a an admixture of the present inventionmay comprise a buffered hydrogel in admixture with a hydrophobiccomposition comprising at least one hydrophobic base and an API. The APImay comprise a NO-releasing API. The hydrophobic composition may furthercomprise one or more of an amphiphilic compound, a cosolvent, ahumectant, and any combination thereof. In certain embodiments, theadmixture may be formed by mixing. In some embodiments, the admixture isself-emulsifying. The admixture may comprise a single phase.

According to embodiments of the present invention, a kit may beprovided. In some embodiments, the kit may comprise a first compositionand a second composition. The first composition may comprise ahydrophilic composition. The second composition may comprise an API,such as, but not limited to, a NO releasing API. In some embodiments,the second composition may comprise at least one hydrophobic base. Inparticular embodiments, the second composition comprises an ointment asdescribed herein and/or such as those described in InternationalApplication Nos. PCT/US2010/046173 and PCT/US2013/028223, which areincorporated herein by reference in their entirety.

In some embodiments, a kit may comprise a first composition and a secondcomposition that are separately stored. In some embodiments, a kit ofthe present invention may comprise means for forming an admixture withthe first composition and second composition, such as, but not limitedto, by mixing, combining, contacting, and the like the compositionsprior to application to a subject. A kit may be configured to admix thetwo compositions upon dispensing and/or for application to a subject. Insome embodiments, a kit may be configured to provide an admixture withincreased performance and/or activity of the API compared to theperformance and/or activity of the API in the absence of one or more ofthe compositions in the admixture.

In use, an admixture may be formed with a first composition and a secondcomposition and then applied to the skin of a subject, and, in someembodiments, including mucosa. For example, the admixture may betopically administered to one or more of a subject's hand, finger, foot,toe, arm, leg, trunk, anus, genitals, face, a mucous membrane (includinga body cavity), nail, etc. In other embodiments, at least onecomposition in the kit may be applied to the skin of a subject and thenat least one different composition in the kit may be applied to sameskin of the subject.

In some embodiments, the admixture comprises a first compositioncomprising a hydrophilic composition and a second composition comprisinga hydrophobic composition. The ratio of the hydrophilic composition tothe hydrophobic composition may be about 5:1 or less, in furtherembodiments, about 4:1 or less, about 3:1 or less, about 2:1 or less orabout 1:1. In certain embodiments, the ratio is about 3:1. In furtherembodiments, the ratio is about 1:1. The admixture may be applied to asubject in such a ratio. In certain embodiments, a kit of the presentinvention comprises means for dispensing and/or delivering the first andsecond compositions in the appropriate amounts to achieve the desiredratio. In some embodiments, the ratio of the first composition andsecond composition in the admixture may be adjusted and/or modified toachieve a desired API release pattern.

Providing a hydrophilic composition and a hydrophobic composition thatare admixed upon application to the skin of a subject may allow for alonger shelf life of a kit of the present invention than if thecompositions were stored and/or mixed together in the kit. For example,the formulation and loading of an API in a hydrophobic composition mayprovide a stable product with a long shelf life. Thus, for example, pHand/or water content of the hydrophobic composition may be adjusted toreduce or minimize release of the API, such as a water activated API, soas to provide a composition that is stable at room temperature. Thehydrophilic composition may then be combined with the hydrophobiccomposition to adjust the combined pH and/or provide water to activatethe API. The hydrophobic composition may be combined with thehydrophilic composition in differing ratios to provide a desiredrelease, pH and/or dose in the admixture. Such an approach may allow fora single manufacturing process to be utilized for production of a morecomplex and costly hydrophobic composition and then particular productsdefined by the composition and/or quantity of the hydrophiliccomposition with which the hydrophobic composition is mixed.

As used herein, the term “shelf life” refers to the length of time aproduct (e.g., a composition and/or kit of the present invention)maintains the ability to release a therapeutically effective amount ofan API, such as, but not limited to, nitric oxide, in an unopenedpackage stored under recommended storage conditions. The shelf life may,for example, be evidenced by the “use by” or “best if used by” date forthe product, the manufacturer's expiration date of the product and/orthe actual product characteristics after the specified period of time.Accordingly, the term “shelf life” as used herein should be construed asincluding both an “actual” shelf life of the product and a “predicted”shelf life of the product unless stated otherwise. As one skilled in theart will recognize, the rate of release of nitric oxide in a compositionunder packaged and/or stored conditions may be different (i.e., fasteror slower) than the rate of release of nitric oxide when the compositionis in use (e.g., when the composition comprising the NO-releasing API isin admixture with another composition). In certain embodiments, the rateof release of nitric oxide from a composition of the present inventionmay be more rapid when the composition is in use compared to the rate ofrelease of nitric oxide when a composition comprising the API waspackaged and/or stored.

In some embodiments, the shelf life of the product is the time that theproduct maintains the ability to release at least 50% of the initialamount of nitric oxide that the product may release when packaged. Infurther embodiments, the shelf life of the product is the time that theproduct maintains the ability to release at least 70%, at least 80%, atleast 90%, at least 95%, or at least 98% of the initial amount of nitricoxide that the product may release when packaged. In some embodiments,the shelf life of the product is the time that the product maintains theability to release a therapeutically effective amount of nitric oxideover a desired period of time. In some embodiments, the recommendedstorage conditions are room temperature. In some embodiments, therecommended storage conditions are refrigerated storage conditions. Inparticular embodiments, the refrigerated storage conditions are between1° C.-12° C., or any range and/or individual value therein.

Further embodiments may provide packaged compositions of the presentinvention that have a useful life of at least about 7 days after openingthe package. In further embodiments, the useful life is at least about30 days, at least about 60 days or at least about 90 days. In stillfurther embodiments, the packaged compositions have a useful life offrom at least about 60 days to at least about 730 days. As used herein,the term “useful life” refers to the length of time that the productmaintains the ability to release a therapeutically effective amount ofnitric oxide from an opened packaged when applied as recommended andwhen stored under recommended storage conditions. The useful life may,for example, be evidenced by the manufacturer's recommended time todispose of the product after opening or measurements of the productscharacteristics after opening.

Accordingly, the term “useful life” as used herein should be construedas including both an “actual” useful life of the product or a“predicted” useful life of the product unless stated otherwise. In someembodiments, the useful life of the product is the time that the productmaintains the ability to release at least 50% of the initial amountnitric oxide that the product may release when the package is opened. Infurther embodiments, the useful life of the product is the time that theproduct maintains the ability to release at least 70%, at least 80%, atleast 90%, at least 95%, or at least 98% of the initial amount nitricoxide that the product may release when the package is opened. In someembodiments, the recommended storage conditions after opening are roomtemperature. In particular embodiments, the recommended storageconditions after opening are refrigerated conditions.

As will be appreciated by those of skill in the art in light of thepresent disclosure, a hydrophilic composition, such as those describedherein, may provide means for adjusting the pH of a pharmaceuticalcomposition as well as means for activating an API of a pharmaceuticalcomposition. In some embodiments, a hydrogel, such as those describedherein, may provide means for maintaining and/or stabilizing the pH of ahydrophobic composition when used to form an admixture with thehydrogel. Means for maintaining and/or stabilizing the pH of anadmixture may be configured to activate and/or initiate release of anAPI. In particular embodiments, a hydrogel of the present invention mayprovide means for maintaining and/or stabilizing the pH of an admixturecomprising a diazeniumdiolate modified co-condensed polysiloxanemacromolecule. In some embodiments, the pH may be maintained and/orstabilized within a pH range of about 5 to about 8. In furtherembodiments, a hydrogel of the present invention may provide means forreleasing nitric oxide from a pharmaceutical composition comprising adiazeniumdiolate modified co-condensed polysiloxane macromolecule.

According to some embodiments, a method of the present inventioncomprises administering a composition of the present invention to theskin of a subject, including mucosa. For example, the composition may beadministered to one or more of a subject's hand, finger, foot, toe, arm,leg, trunk, anus, genitals, face, a mucous membrane (including a bodycavity), nail, etc. In certain embodiments, the composition may betopically administered. In some embodiments, a hydrophilic compositionof the present invention may be topically administered to the skin of asubject. In certain embodiments, an admixture comprising a hydrophobiccomposition and a hydrophilic composition may be topically administeredto the skin of a subject. The admixture may comprise at least one API,such as, but not limited to, a NO-releasing API.

A method of the present invention may comprise forming an admixtureprior to and/or during the administering step. An admixture may beprepared by mixing, blending, contacting, applying to a same area orregion, emulsifying, and the like a hydrophilic composition such as, butnot limited to, a hydrogel, and a hydrophobic component such as, but notlimited to an ointment.

In some embodiments, a method of the present invention comprisesdelivering a therapeutically effective amount of nitric oxide to theskin of a subject. As used herein, the term “therapeutically effectiveamount” refers to an amount of an API, such as, but not limited to,nitric oxide, that elicits a therapeutically useful response in asubject Those skilled in the art will appreciate that the therapeuticeffects need not be complete or curative, as long as some benefit isprovided to the subject.

The present invention finds use in both veterinary and medicalapplications. Subjects suitable to be treated with a method embodimentof the invention include, but are not limited to, avian and mammaliansubjects. Mammals of the present invention include, but are not limitedto, canines, felines, bovines, caprines, equines, ovines, porcines,rodents (e.g. rats and mice), lagomorphs, primates (e.g., simians andhumans), non-human primates (e.g., monkeys, baboons, chimpanzees,gorillas), and the like, and mammals in utero. Any mammalian subject inneed of being treated according to the present invention is suitable.Human subjects of both genders and at any stage of development (i.e.,neonate, infant, juvenile, adolescent, adult) may be treated accordingto the present invention. In some embodiments of the present invention,the subject is a mammal and in certain embodiments the subject is ahuman. Human subjects include both males and females of all agesincluding fetal, neonatal, infant, juvenile, adolescent, adult, andgeriatric subjects as well as pregnant subjects. In particularembodiments of the present invention, the subject is a human adolescentand/or adult.

Illustrative avians according to the present invention include chickens,ducks, turkeys, geese, quail, pheasant, ratites (e.g., ostrich) anddomesticated birds (e.g., parrots and canaries), and birds in ovo.

The methods of the present invention may also be carried out on animalsubjects, particularly mammalian subjects such as mice, rats, dogs,cats, livestock and horses for veterinary purposes, and/or for drugscreening and drug development purposes.

In particular embodiments of the present invention, the subject is “inneed of” a method of the present invention, e.g., the subject has beendiagnosed with, is at risk for, and/or is believed to have a disease ordisorder that may be treated using a method of the present invention. Insome embodiments, the subject has a skin disorder, such as, but notlimited to, acne, atopic dermatitis, and/or psoriasis. In otherembodiments, the subject has a wound, such as, but not limited to, a bedsore, a burn, a chronic venous leg ulcer, and/or a diabetic foot ulcer.In some embodiments of the present invention, the subject has aninflammatory skin condition or disorder. In some embodiments of thepresent invention, the subject has an infection, such as a viral,bacterial or fungal infection and, in particular embodiments, aninfection with a cutaneous symptom. In some embodiments, the subject hasa cosmetic condition, such as a scar, crow's feet, etc. In still furtherembodiments, the subject has a cancer of the skin.

“Treat,” “treating” or “treatment of” (and grammatical variationsthereof) as used herein refer to any type of treatment that imparts abenefit to a subject and may mean that the severity of the subject'scondition is reduced, at least partially improved or ameliorated and/orthat some alleviation, mitigation or decrease in at least one clinicalsymptom is achieved and/or there is a delay in the progression of thedisease or disorder. In particular embodiments, the severity of a skindisorder may be reduced in a subject compared to the severity of theskin disorder in the absence of a method of the present invention. Inother embodiments, a method of the present invention may improve woundhealing and/or prevent against infection.

A composition of the present invention may be applied topically to anyportion of a subject's skin. However, in some embodiments, the subject'sface is treated by a method described herein. Furthermore, in someembodiments, the subject's trunk is treated by a method describedherein. In certain embodiments, a composition of the present inventionis applied to a wound present on a subject.

According to some embodiments, a method of increasing the release ofnitric oxide from a hydrophobic composition containing adiazeniumdiolate modified macromolecule may be provided. The method maycomprise forming an admixture; and applying the admixture to the skin ofa subject. The admixture may comprise at least one hydrophiliccomposition and at least one hydrophobic composition comprising thediazeniumdiolate modified macromolecule. In some embodiments, thehydrophilic composition may have a pH of about 4 to about 6. The formingstep or admixing step may be carried out on the skin of the subject ormay be carried out prior to application of the admixture to the skin ofthe subject.

A method of the present invention may increase the amount of nitricoxide released by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90, 100%, 150%, 200%, 300%, 400%, or more, or any range and/orindividual value therein compared to the amount of NO released in theabsence of a method of the present invention over the same period oftime. A method of the present invention may provide a NO release that isincreased by about 1.5 to about 100 times than the amount of NO releasedin the absence of a method of the present invention over the same periodof time or any range and/or individual value therein, such as, but notlimited to by about 2 to about 10 times or by about 5 to about 50 times.

In further embodiments, a method of providing a topical antimicrobialcomposition may be provided. The method may comprise forming anadmixture; and applying the admixture to the skin of a subject. Theadmixture may comprise at least one hydrophilic composition and at leastone hydrophobic composition comprising a diazeniumdiolate modifiedmacromolecule. In some embodiments, the hydrophilic composition may havea pH of about 4 to about 6. The forming step or admixing step may becarried out on the skin of the subject or may carried out prior toapplication of the admixture to the skin of the subject. A method of thepresent invention may inhibit the growth of a pathogen, such as by about5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90, 100%, 150%, 200%, ormore compared to the growth of a pathogen in the absence of a method ofthe present invention.

A method of increasing the rate of healing for a wound may also beprovided. The method may comprise applying topically an admixture of thepresent invention to a wound. The admixture may comprise at least onehydrophilic composition and at least one hydrophobic compositioncomprising the diazeniumdiolate modified macromolecule. In someembodiments, the hydrophilic composition may have a pH of about 4 toabout 6. The admxiture may be antimicrobial and/or may be configured tobuffer the wound to a pH below 7. In some embodiments, a method of thepresent invention may increase the rate of healing for a wound by atleast about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90, 100%, 150%,200%, or more compared to the rate of healing from a similar wound inthe absence of a method of the present invention. In some embodiments, amethod of the present invention may increase tissue oxygen availabilityand/or reduce the histotoxicity of a bacterial end product.

A composition, kit, and/or method of the present invention may minimizeand/or prevent degradation of at least one API such as, but not limitedto, a NO-releasing API. In some embodiments, a composition of thepresent invention may be configured to provide a repeatable rate and/orpattern of NO release to a variety of therapeutic sites with varyingmoisture contents. In certain embodiments, a composition of the presentinvention may be configured to provide a rate of NO release that isindependent of the moisture content present at a therapeutic site.

The present invention is explained in greater detail in the followingnon-limiting Examples.

EXAMPLES Example 1

An in vitro test comparing the release of nitric oxide from an ointmentin combination with different hydrophilic phases was performed. Theointment was combined with either wet nitrogen (i.e., neat), water in a1:2 ratio (ointment:water) or a hydrogel in a 1:1 ratio. The formulationfor the ointment and hydrogel are provided in Table 1.

TABLE 1 Ointment and hydrogel formulation. Ointment % Hydrogel %Crodabase SQ, 55.5 Anhydrous Glycerol 10.0 Mineral Oil/PolyethyleneLight Mineral oil 13.5 Triethanolamine 1.2 Miglyol 812 12.0 Carbopol ®974P, 0.6 Caprylic/Capric Carbomer Homopolymer Triglyceride Type ASoftigen 767 10.0 Deionized Water 88.2 PEG-6-Caprylic/Capric GlyceridesHexylene glycol 8.0 Nitricil ™ NVN1 1.0 Total 100 Total 100

The in vitro test of the nitric oxide release showed a significantdifference before and after addition of the hydrophilic phase even incarrier gas with full moisture content. The hydrophilic phase's abilityto promote the nitric oxide release from the hydrophobic phase,especially at a lower Nitricil™ NVN1 loading, is significantly higherthan water alone. While not wishing to be bound to any particulartheory, the results suggest that the efficiency to provide access ofprotons to the drug substance in the hydrophobic phase increasesremarkably with the excipients of emollient and solubilizing capacities(Table 2).

TABLE 2 Nitric oxide release from a 1% Nitricil ™ NVN1 ointment incombination with different hydrophilic phases. Parameter Wet N₂ WaterHydrogel Ointment Sample Weight (mg) 52.0 44.2 46.2 Cmax (ppb) 307.02,600.0 13,700.0 Tmax (min) 6.7 30.0 1.1 Total Nitric Oxide Release976,055.0 4,385,884.0 14,926,266.0 in First Hour (PPB) Total NitricOxide Release 18,770.0 99,228.0 323,079.4 in First Hour per SampleWeight (PPB/mg)

The results demonstrate that the hydrogel works well in promoting nitricoxide release from the hydrophobic ointment with lower Nitricil™ NVN1loading. However, when the potency for the drug product increases, therelease kinetics do not follow the loading capacity (i.e., the nitricoxide release does not increase proportionally with the percentage ofthe drug substance). The sluggish release becomes more significant withthe higher drug substance loading over 10% by weight. While not wishingto be bound to any particular theory, it is believed that the pH of thefinal formulation is too high for the higher loading drug product torelease effectively.

Example 2

It was discovered by the present inventors that in order to maintain acontrolled release of nitric oxide from the drug product at differentloadings, the concentration of the key reagent, proton, needs to bemaintained. This means that the final pH of a composition comprising ahydrophilic phase (e.g., a hydrogel) and a hydrophobic phase (e.g., anointment) needs to stay within a specified range across differentpotencies. To achieve this, the hydrophilic phase of the composition canbe designed to have a larger buffer capacity than the hydrophobic phaseat the highest target drug product potency.

In order to have both a large buffer capacity and a desired range of thefinal formulation at pH 5-8, phosphoric acid monobasic was selected as abuffer for the hydrogel. For the [H₂PO₄]⁻ ion, the pKa is 7.2. Accordingto the Henderson-Hasselbalch equation (Equation 1), the pH of thesolution at an equal concentration of the acid and base will be in theoptimal neutral condition as the drug product, (pH=7.2), giving it alarge buffer capacity.

$\begin{matrix}{{{Henderson}\text{-}{Hasselbalch}\mspace{14mu} {{equation}.{pH}}} = {{pK}_{a} + {\log \left( \frac{\left\lbrack A^{-} \right\rbrack}{\lbrack{HA}\rbrack} \right)}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

A hydrogel with 400 mmol of phosphoric acid monobasic (i.e., phosphatebuffered hydrogel) and having a pH value of 4.8(+/−0.1) was prepared(Table 3). The hydrogel was then mixed with a Nitricil™ NVN4 ointmenthaving a 0.9% weight of nitric oxide loading to determine the nitricoxide release results. Table 3 provides the formulations for theointment and hydrogel.

TABLE 3 Ointment and hydrogel formulations. Ointment % PhosphateBuffered Hydrogel % Crodabase SQ, 55.9 Water, deionized 71.8 MineralOil/Polyethylene Light Mineral oil 4.7 Potassium phosphate 5.2 monobasicMiglyol 812 11.9 Hexylene glycol 19 Caprylic/Capric TriglycerideSoftigen 767 9.9 Hydroxyethyl cellulose 3 PEG-6-Caprylic/Capricethoxylate, quaternized Glycerides Hexylene glycol 7.9 Nitricil ™ NVN49.7 Total 100 Total 100

When the phosphate buffered hydrogel was mixed with the ointment, the pHincreased, but to a much lesser degree compared to the hydrogelformulation without phosphate described in Example 1. Table 4 providesthe in vitro nitric oxide release results of combined ointment/phosphatebuffered hydrogel at three different NO loadings. The combinedointment/phosphate buffered hydrogel is compared to two separatecombinations of the ointment with one of two non-buffered hydrogelformulations at different pH values. The non-buffered hydrogelformulations have a composition as set forth in Table 1 and a pH of 4and 6, respectively. The results clearly show that the phosphatebuffered hydrogel can stabilize the final formulation pH at the desiredrange, and promote high levels of nitric oxide release across all threeloadings.

TABLE 4 In vitro nitric oxide release and pH data for Nitricil ™ NVN4ointment admixtures. Cumulative C_(max) NO Ointment Hydrophilic Phasepmol/mg nmol/mg pH 3.2% Phosphate Buffered 30 54 6.1 Nitricil ™ HydrogelpH 4.8 NVN4 (Phogel48) (0.3% NO) Nonbuffered pH 4 13 39 6.1 (AH-002)Nonbuffered pH 6 9.8 16 9.1 (AH-001) 9.7% Phosphate Buffered 30 108 6.9Nitricil ™ Hydrogel pH 4.8 NVN4 (Phogel48) (0.9% NO) Nonbuffered pH 46.6 48 9.0 (AH-002) Nonbuffered pH 6 2.6 23.2 9.9 (AH-001) 19.4%Phosphate Buffered 13 114 7.9 Nitricil ™ Hydrogel pH 4.8 NVN4 (Phogel48)(1.8% NO) Nonbuffered pH 4 6.8 62.0 9.5 (AH-002) Nonbuffered pH 6 5.925.5 10.1 (AH-001)

Example 3

A comparison of the nitric oxide release from a Nitricil™ NVN4 ointmentwith a 1.8% NO loading in the absence of and with different moisturesources (i.e., hydrophilic phases) was performed. The ointmentformulation is provided in Table 3. Two different moisture sources werecombined with the ointment. The first source was a neutral hydrogel witha pH of 6 and having a formulation as described in Example 1, Table 1,and the second was a phosphate buffered hydrogel having a formulation asdescribed in Example 2, Table 3. FIG. 1 shows the NO release from theointment alone and the NO release the ointment/hydrophilic phaseadmixtures, and the results are shown in Table 5.

TABLE 5 NO release parameters for different formulations containingNitricil ™ NVN4 ointment (1.8% w/w NO). Hydrophobic Hydrophilic C_(max)CumulativeNO T₅₀ Phase Phase (pmol/mg) (nmol/mg) (min) Ointment N/A 0.5529 777 Ointment Neutral 1.6 95 609 Hydrogel (AH-001) Ointment Phosphate15.2 191 421 Buffered Hydrogel (Phogel48)

Example 4

Three Nitricil™ NVN4 ointment formulations having different NO loadingswere each combined with a phosphate buffered hydrogel in a 1:1 ratio toform an admixture. The formulations for the ointment and hydrogel areprovided in Table 6.

TABLE 6 Ointment and hydrogel formulations. Ointment FormulationHydrogel Formulation (TO-007) (Phogel48) Component 3.2% 9.7% 19.4%Component % Crodabase SQ, 61.1 55.9 48.9 Water, 71.8 Mineral Oil/deionized Polyethylene Light Mineral oil 6.0 4.7 2.0 Potassium 5.2phosphate monobasic Miglyol 812 11.9 11.9 11.9 Hexylene glycol 19Caprylic/Capric Triglyceride Softigen 767 9.9 9.9 9.9 Hydroxyethyl 3.0PEG-6-Caprylic/ cellulose ethoxy- Capric Glycerides late, quaternizedHexylene glycol 7.9 7.9 7.9 Nitricil ™ NVN4 3.2 9.7 9.7 Total 100 100100 Total 100

The vehicle ointment did not contain the drug product, Nitricil™ NVN4,but contained all other components in the Nitricil™ NVN4 ointmentformulations. Mupirocin was used as a positive control. Theseformulations (i.e., the test articles) were then tested for efficacyagainst MRSA Staphylococcus aureus in a porcine animal model. ATCC BAA1686 bacterial counts were taken on days 4 and 7 after treatmentapplication.

Three specific pathogen-free pigs (Looper Farms, NC) were anesthetizedand 51 rectangular wounds (10 mm×7 mm×0.5 mm deep) were made to theparavertebral and thoracic area with an elecrokeratome. Wounds wereseparated by 15 mm of unwounded skin and individually dressed. Eightwounds were randomly assigned to each treatment group (6) and baseline.A fresh culture of S. aureus strain BAA-1686 was prepared from a TSAplate grown overnight at 37° C. S. aureus from the overnight culture wasresuspended in 4.5 ml of saline until a solution corresponding to 10¹⁰CFU/ml was obtained. Serial dilution was performed to create an initialinoculum concentration of 10⁶ CFU/ml was achieved. 25 μl of the inoculumsuspension was inoculated into each wound by scrubbing with a sterilespatula for 10 seconds. All wounds were covered individually with apolyurethane film dressing (TEGADERM™). The bacterial biofilms wereallowed to form for 48 hours prior to treatment. Treatment groups weretreated with approximately 200 mg of test article and spread out tocover the wound and surrounding unwounded area with a sterile spatulaand covered with film dressing. At the assessment time, 4 wounds pertreatment group were recovered in 1 ml of neutralization solution andserially diluted. Serial dilutions were subsequently plated on OxacillinResistance Screening Agar (ORSA) and incubated for 24 hours at 37° C.prior to enumeration of viable colonies. The MRSA BAA-1686 bacterialcounts following treatment are provided in FIGS. 2 and 3.

While not wishing to be bound to any particular theory, the data suggestthat Nitricil™ macromolecules exhibit robust antimicrobial activityagainst biofilm-embedded S. aureus. Unlike traditional antibiotics,Nitricil™ NVN4 was effective at reducing biofilm populations. Nitricil™macromolecules may be an effective therapy for the treatment of chronicS. aureus infections and may be an efficacious antimicrobial agent invivo using a partial thickness wound model.

Example 5

Ointment formulations were prepared as follows in Tables 7 and 8, whichlist the percent of each component by weight of the respective ointmentformulation. Buffered hydrogel formulations were prepared as follows inTable 9, which lists the percent of each component by weight of therespective buffered hydrogel formulation and pH of each bufferedhydrogel formulation.

TABLE 7 Composition of example ointment formulations. Component TO-008TO-009 TO-010 TO-011 TO-012 TO-013 TO-014 TO-015 TO-016 TO-018 PEG400 —89.0  — — — 20.0 20.0 — 38.0 — Crodabase SQ 60.0 — 79.0  60.0 60.0 60.060.0 60.0 60.0 53.0 Petrolatum, — — — — — — — — — 15.0 White LightMineral 13.0 — 8.0 11.0 11.0 13.0 13.0 — — — Oil Mineral Oil — — — — — —— 13.0 — 10.0 Miglyol ® 812 12.0 — 6.0 12.0 12.0 — — 12.0 — 12.0Miglyol ® 840 — — — — 10.0 — — — — — Cyclomethicone — — — 10.0 — — — — —— PEG 3350 — 9.0 — — — — — — — — Hexylene Glycol  8.0 — — — — — —  8.0 —— Cetyl Alcohol — — — — — — — — —  8.0 Softigen ® 767  5.0 — 5.0  5.0 5.0 —  5.0  5.0 —  2.0 Nitricil ™ NVN1  2.0 2.0 2.0  2.0  2.0  2.0  2.0 2.0  2.0  2.0

TABLE 8 Composition of further example ointment formulations. TO-017Component A B C D E F G H I J Crodabase SQ — 26.7 40.0 13.3 0.00 80.013.3 40.0 53.4 — Petrolatum, — 26.7 40.0 13.3 40.0 — 53.4 — 13.3 80.0White Cetyl Alcohol 80.0 26.6 — 53.4 40.0 — 13.3 40.0 13.3 — Miglyol ®812 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0 Mineral Oil 4.04.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 Softigen ® 767 2.0 2.0 2.0 2.0 2.02.0 2.0 2.0 2.0 2.0 Nitricil ™ NVN1 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.02.0

TABLE 9 Composition of example buffered hydrogel formulations.Unbuffered Buffered AH-010 CA-001 CA-002 CA-003 PHO-002 PHO-003 PHO-004Component (pH 6) (pH 5.5) (pH 6.5) (pH 5.5) (pH 5) (pH 6) (pH 7)Purified Water 87.1  83.0  83.0  83.7  80.6 85.0 73.5 Glycerin 10.0 10.0  10.0  10.0  10.0 10.0 10.0 Potassium Phosphate — — — —  5.9 1.35.8 Monobasic Potassium Phosphate — — — — — 0.2 7.2 DibasicCarboxymethylcellulose 2.8 4.0 — 1.5  3.5 3.5 3.5 Sodium Sodium Alginate— — 4.0 — — — — Citric Acid, Anhydrous — 2.0 1.8 1.8 — — — SodiumHydroxide — 0.8 1.0 1.0 — — — Phenoxyethanol — — — 0.6 — — — Trolamine —— — 0.6 — — — Carbopol ® 974P — — — 0.4 — — — Sorbic Acid — 0.2 — — — —— Benzoic Acid 0.1 Methylparaben — — 0.1 0.2 — — — Propylparaben — 0.1 —— — — Total 100.0  100.0  100.0  100.0  100.0  100.0 100.0

Example 6

Three specific pathogen-free pigs (Looper Farms, NC) per microorganismwere anesthetized and 51 rectangular wounds (10 mm×7 mm×0.5 mm deep)were made to the paravertebral and thoracic area with an elecrokeratome.Wounds were separated by 15 mm of unwounded skin and individuallydressed. Eight wounds were randomly assigned to each treatment group andbaseline. After creation of the burns/wounds, 25 μl of Acinetobacterbaumannii (AB 09-001*), Methicillin Resistant S. aureus (MRSA USA300)and Candida albicans (CA 09-024*) were use to inoculate each wound byscrubbing (10⁶ CFU/ml) inoculums into each wound with a teflon spatulafor approximately 30 seconds. All wounds were covered individually witha polyurethane film dressing (TEGADERM™). The bacterial biofilms wereallowed to form for 48 hours prior to treatment.

Treatment groups were treated with approximately 200 mg of test articleand spread out to cover the wound and surrounding unwounded area with asterile spatula and covered with the film dressing. The test articlesinclude the three Nitricil™ NVN4 ointment formulations having differentNO loadings described in Table 6 of Example 4, which were each combinedwith the phosphate buffered hydrogel described in Table 6 of Example 4.The vehicle ointment did not contain the drug product, Nitricil™ NVN4,but contained all other components in the Nitricil™ NVN4 ointmentformulations. Silver sulfadiazine was used as a positive control.

At the assessment time, 4 wounds per treatment group were recovered in 1ml of neutralization solution and serially diluted. Serial dilutionswere subsequently plated on selective media and incubated for 24 hoursat 37° C. prior to enumeration of viable colonies. Colonies were countedand the colony forming units per ml (CFU/ml), Log CFU/ml, mean LogCFU/ml and standard deviation calculated. A one-way analysis of variance(ANOVA) was used for statistical analysis. A p value of less than 0.05was considered significant. FIG. 4 shows the results for A. baumannii,FIG. 5 shows the results for S. aureus, and FIG. 6 shows the results forC. albicans.

While not wishing to be bound to any particular theory, the data suggestthat Nitricil™ macromolecules are an effective antibacterial againstbiofilm-embedded A. baumannii, S. aureus, and C. albicans. Thus,Nitricil™ macromolecules may be an effective therapy for the treatmentof chronic A. baumannii, S. aureus, and C. albicans infections and maybe an efficacious antimicrobial agent in vivo using a partial thicknesswound model.

Example 7

The release of nitric oxide from three different admixtures wascompared. The admixtures were formed with an ointment formulation,TO-007b as provided in Table 10, having varying concentrations ofNitricil™ NVN4 and a hydrogel formulation, Phogel48 as provided in Table6 of Example 4. The ointment formulations had 3.2% Nitricil™ NVN4 (0.3%NO content), 6.4% Nitricil™ NVN4 (0.9% NO content), or 12.8% Nitricil™NVN4 (1.8% NO content) and were combined with the hydrogel in a 1:1ratio.

TABLE 10 Ointment formulation. Ointment Formulations (TO-007b) 3.2% 6.4%12.8% Nitricil ™ Nitricil ™ Nitricil ™ Component NVN4 NVN4 NVN4Crodabase SQ 60.0 60.0 58.0 Miglyo ®l 812 12.0 12.0 12.0 Hexylene glycol8.0 8.0 8.0 Softigen ® 767 5.0 5.0 5.0 Light Mineral Oil 11.8 8.6 4.2Nitricil ™ NVN4 3.2 6.4 12.8 Total 100 100 100

The real time NO release and cumulative NO for the different admixturesare provided in Table 11. The cumulative NO release over time for theadmixtures is provided in FIG. 7 and the real-time concentration of NOrelease over time for the admixtures is provided in FIG. 8.

TABLE 11 Admixture NO release properties. Real-Time NO ReleaseCumulative NO Time (pmol/mg) Time (nmol/mg) (hr) 3.2% 6.4% 12.8% (hr)3.2% 6.4% 12.8% 0.50 7.15 17.63 7.89 0.50 28.13 46.90 15.33 1.0 2.037.47 6.18 1.0 35.39 66.88 27.80 2.0 0.49 2.76 4.42 2.0 38.95 85.29 45.473.0 0.29 1.80 2.94 3.0 40.09 93.60 57.89 4.0 0.17 1.15 2.34 4.0 40.8998.89 67.19 8.0 0.00 0.48 1.48 8.0 42.55 109.88 93.19 12.0 0.93 12.0110.52 18.0 0.11 18.0 121.81

Example 8

The release of nitric oxide from two different ointments was compared.The ointment formulation was TO-007b, as provided in Table 12,containing either 12.8% Nitricil™ NVN4 (1.8% NO content) or 2% Nitricil™NVN1 (0.3% NO content). The real time NO release and cumulative NO forthe different ointments are provided in Table 13. The cumulative NOrelease over time for the admixtures is provided in FIG. 9 and thereal-time concentration of NO release over time for the admixtures isprovided in FIG. 10.

TABLE 12 Ointment formulations. Ointment Formulations (TO-007b) 2% 12.8%Nitricil ™ Nitricil ™ Component NVN1 NVN4 Crodabase SQ 60.0 58.0Miglyol ® 812 12.0 12.0 Hexylene glycol 8.0 8.0 Softigen ® 767 5.0 5.0Light Mineral Oil 13.0 4.2 Nitricil ™ NVN1 2.0 — Nitricil ™ NVN4 — 12.8Total 100 100

TABLE 13 Ointment NO release properties. Real-Time NO Release CumulativeNO (pmol/mg) (nmol/mg) Time 2% 12.8% Time 2% 12.8% (hr) NVN1 NVN4 (hr)NVN1 NVN4 0.50 0.53 0.35 0.50 0.75 0.40 1.00 0.67 0.35 1.00 1.86 1.062.00 0.63 0.26 2.00 4.29 2.17 3.00 0.59 0.24 3.00 6.48 3.03 4.00 0.500.21 4.00 8.38 3.92 8.00 0.33 8.00 7.89 12.00 0.40 12.00 13.26 18.000.46 18.00 22.51 21.50 0.50 21.50 28.54

1. A topical pharmaceutical composition comprising a nitricoxide-releasing active pharmaceutical ingredient in admixture with ahydrophilic composition and a hydrophobic composition, wherein thehydrophobic composition comprises the nitric oxide-releasing activepharmaceutical ingredient and the nitric oxide-releasing activepharmaceutical ingredient comprises a nitric oxide-releasing compoundhaving a diazeniumdiolate functional group.
 2. The topicalpharmaceutical composition of claim 1, wherein the hydrophiliccomposition comprises a solution, suspension, lotion, gel, cream, orhydrogel.
 3. (canceled)
 4. The topical pharmaceutical composition ofclaim 1, wherein the hydrophilic composition has a pH of about 3 toabout
 8. 5. The topical pharmaceutical composition of claim 1, whereinthe hydrophilic composition comprises a buffer, optionally a phosphatebuffer.
 6. The topical pharmaceutical composition of claim 1, whereinthe hydrophilic composition comprises a polymer, optionally cellulose ora pharmaceutically acceptable salt thereof, chitosan, an acrylic acidpolymer, or any combination thereof.
 7. The topical pharmaceuticalcomposition of claim 1, wherein the hydrophilic composition comprises apolyhydric alcohol. 8-9. (canceled)
 10. The topical pharmaceuticalcomposition of claim 1, wherein the topical pharmaceutical compositionis self-emulsifying.
 11. The topical pharmaceutical composition of claim1, wherein the composition has a viscosity of about 5000 cP to about100,000 cP.
 12. (canceled)
 13. The topical pharmaceutical composition ofclaim 1, wherein the topical pharmaceutical composition is continuous.14. The topical pharmaceutical composition of claim 1, wherein thenitric oxide-releasing compound comprises a NO-releasing co-condensedsilica particle.
 15. The topical pharmaceutical composition of claim 1,wherein the topical pharmaceutical composition has a pH of about 3 toabout
 9. 16. The topical composition of claim 1, wherein the compositionfurther comprises a hydrophobic base; at least one of an amphiphiliccompound or an emulsifying agent; a buffer; a polymer; a polyhydricalcohol; and water, and wherein the composition is buffered to a pH ofabout 3 to about
 9. 17. The topical pharmaceutical composition of claim16, wherein the nitric oxide-releasing active pharmaceutical ingredient,the hydrophobic base, and the at least one of the amphiphilic compoundor the emulsifying agent are present in the hydrophobic composition andthe buffer, the polymer, the polyhydric alcohol, and the water arepresent in the hydrophilic composition. 18-34. (canceled)
 35. A kitcomprising: a first composition comprising a hydrophilic composition;and a second composition comprising an active pharmaceutical ingredient,wherein the active pharmaceutical ingredient comprises a nitricoxide-releasing compound having a diazeniumdiolate functional group. 36.The kit of claim 35, wherein the hydrophilic composition has a pH ofabout 4 to about
 8. 37. The kit of claim 35, wherein the hydrophiliccomposition is a hydrogel, optionally a buffered hydrogel.
 38. The kitof claim 35, wherein the hydrophilic composition comprises: a phosphatebuffer; a cellulose or a pharmaceutically acceptable salt thereof; apolyhydric alcohol; and water.
 39. The kit of claim 35, wherein thesecond composition further comprises: a hydrophobic base; and anamphiphilic compound.
 40. The kit of claim 35, wherein the firstcomposition and second composition are separately stored.
 41. A methodof stably storing a diazeniumdiolate modified macromolecule in ahydrophobic composition and releasing nitric oxide from thediazeniumdiolate modified macromolecule, the method comprising: admixingthe hydrophobic composition with a hydrophilic composition to form anadmixture, wherein the hydrophobic composition comprises thediazeniumdiolate modified macromolecule and the hydrophilic compositionhas a pH of about 4 to about
 8. 42-48. (canceled)